Pyrazine derivatives

ABSTRACT

The invention concerns pyrazine derivatives of the Formula I 
     
       
         
         
             
             
         
       
     
     or pharmaceutically-acceptable salts thereof; wherein each of n, m and R has any of the meanings defined hereinbefore in the description; processes for their preparation, pharmaceutical compositions containing them and their use in the manufacture of a medicament for use in the treatment of bone-related disorders or conditions.

This application is a continuation of U.S. application Ser. No.13/008,154 (filed Jan. 18, 2011), which claims the benefit under 35U.S.C. §119(e) of U.S. Application No. 61/296,196 (filed Jan. 19, 2010).

The invention concerns certain novel pyrazine derivatives, orpharmaceutically-acceptable salts thereof, which possess bone formationactivity and are accordingly useful in methods of treatment of the humanor animal body. The invention also concerns processes for themanufacture of said pyrazine derivatives, pharmaceutical compositionscontaining said pyrazine derivatives and use of said pyrazinederivatives in therapeutic methods, for example in the manufacture ofmedicaments for use in the prevention or treatment of bone-relateddisorders or conditions in a warm-blooded animal such as man.

The present invention especially relates to pyrazine derivatives thatare inhibitors of Glycogen synthase kinase 3 (GSK3).

GSK3 is a serine/threonine protein kinase composed of two isoforms (αand β), which are encoded by distinct genes but are highly homologouswithin the catalytic domain. GSK3 is highly expressed in the central andperipheral nervous system. GSK3 is a multi-functional kinase thatregulates a number of different signaling pathways involved in importantcellular functions, structure, gene expression, mobility and survival(Jope et al Neurochem Res. 2007, 32, 577-595). GSK3 phosphorylatesseveral substrates including tau, β-catenin, glycogen synthase, pyruvatedehydrogenase and elongation initiation factor 2b (eIF2b). Insulin andgrowth factors activate protein kinase B, which phosphorylates GSK3 onserine 9 residue and inactivates it (Kannoji et al, Expert Opin. Ther.Targets 2008, 12, 1443-1455).

GSK3, is a constitutively active kinase, which is now recognized toregulate pathways including Wnt/β-catenin, Notch and PI3K and hedgehogsignaling pathways and has a role in regulating processes includingosteogenesis, chrondogenesis and adipogenesis (Kulkarni et al., J CellBiochem, 2007, 102, 1504-1518). GSK30, one of the isoforms of GSK3, hasbeen demonstrated to regulate bone formation both in vitro and in vivo.Inhibition of GSK3 prevents the phosphorylation of β-catenin leading toβ-catenin stabilization, accumulation and translocation from cytoplasmto nucleus. This process is thought to lead to activation oftranscription factors and transcription of specific genes involved inbone formation, for example induction of expression of alkalinephosphatase mRNA and protein, a marker of early osteoblastdifferentiation (Bain et al., Biochem. Biophys. Res. Commun., 2003,301:84-91). GSK3 inhibition in murine mesenchymal stem cells inducesmarkers of osteoblast differentiation and bone formation in vitro(Kulkarni et al., JBMR, 2006, 6, 910-920). Inhibition of GSK3β protectsagainst glucocorticoid induced suppression of survival anddifferentiation of osteoblast cultures in vitro (MC3T3-E1 cells, Wang etal. Life Sciences, 2009, 85, 685-692). Heterozygous GSK3β-deficient micedisplayed an increased bone formation (Kugimiya F et al., PLoS One,2007, 2 (9), e837, 1-9). In addition, particular inhibitors of GSK3 havebeen demonstrated to increase bone mass and improve bone strength in anovariectomized animal model of bone loss (Kulkarni et al., JBMR, 2006,21, 910-920) and attenuate glucocorticoid induced bone loss in animals(Wang et al. Life Sciences, 2009, 85, 685-692). In addition, inhibitionof GSK3 prevents myeloma-induced suppression of bone formation and thedevelopment of osteolytic disease in the 5T2MM model of myeloma (Abdulet al., Bone, 2009, 44 (suppl.1), S53 Abstract 103).

Genetic studies have established a link between bone mass in humans andWnt signaling (Gong et al., Am. J. Hum. Genet. 1996, 59, 146-51: Littleet al., N. Engl. J. Med., 2002, 347, 943-4). Genetic and pharmacologicalmanipulations of Wnt signaling in mice have since then confirmed thecentral role of this pathway in regulating bone formation. Of thepathways activated by Wnts, it is signaling through the canonical (i.e.,Wnt/β-catenin) pathway that increases bone mass through a number ofmechanisms including renewal of stem cells, stimulation ofpre-osteoblast replication, induction of osteoblastogenesis, andinhibition of osteoblast and osteocyte apoptosis. Therefore, enhancingWnt pathway signaling with GSK3 inhibitors could be used for thetreatment of bone-related disorders, or other conditions which involve aneed for new and increased bone formation, for example osteoporosis(genetic, iatrogenic or generated through aging/hormone imbalance),fracture repair as a result of injury or surgery, chronic-inflammatorydiseases that result in bone loss such as for example rheumatoidarthritis, cancers that lead to bone lesions, such as for examplecancers of the breast, prostate and lung, multiple myeloma,osteosarcoma, Ewing's sarcoma, chondrosarcoma, chordoma, malignantfibrous histiocytoma of the bone, fibrosarcoma of the bone, cancerinduced bone disease, iatrogenic bone disease, benign bone disease andPaget's disease.

These findings suggest that pharmacological inhibitors of GSK3 enzymesshould be of therapeutic value for treatment of the various forms ofbone-related disorders or conditions, such as, for example osteoporosis(genetic, iatrogenic or generated through aging/hormone imbalance),fracture repair as a result of injury or surgery, chronic-inflammatorydiseases that result in bone loss such as for example rheumatoidarthritis, cancers that lead to bone lesions, such as for examplecancers of the breast, prostate and lung, multiple myeloma,osteosarcoma, Ewing's sarcoma, chondrosarcoma, chordoma, malignantfibrous histiocytoma of the bone, fibrosarcoma of the bone, cancerinduced bone disease, iatrogenic bone disease, benign bone disease andPaget's disease.

In addition to bone-related disorders or conditions, there is evidencethat GSK3 enzymes play a role in other diseases. GSK3 inhibition mayhave beneficial effects in Alzheimer's disease, dementias, andtaupathies, acute neurodegenerative diseases, bipolar disorders,schizophrenia, diabetes, alopecia, inflammatory disease, cancer,glaucoma and regenerative medicine. Thus inhibitors of GSK3 enzymes, areexpected to be of value in the prevention and treatment of a widevariety of diseases in addition to bone-related disorders or conditions.

The compounds, i.e. the pyrazine derivatives, of the invention have nowsurprisingly been found to possess potent bone forming activity, beinguseful in preventing or treating bone-related disorders or conditions.Without wishing to imply that the compounds disclosed in the presentinvention possess pharmacological activity only by virtue of an effecton a single biological process, it is believed that the compoundsprovide a bone forming effect by way of inhibition of GSK3 enzymes.

It is well known that severe problems such as toxicity or reducedefficacy may occur if plasma levels of one drug are altered by theco-administration of another drug. This phenomenon, which is oftencalled drug-drug interactions, could occur if there is a change in themetabolism of one drug caused by the co-administration of anothersubstance possessing liver enzyme inhibiting or inducing properties.Cytochromes P450 are the principal, hepatic xenobiotic metabolizingenzymes and the CYP (cytochrome P450) 3A4 isoform is generally regardedas one of the most important enzymes in the human liver as the majorityof oxidised drugs have been biotransformed by this enzyme. Accordingly,it is undesirable to employ a medication having a significant degree ofsuch liver enzyme inhibiting or inducing properties. This isparticularly important in diseases such as those the compounds of theinvention could be useful to treat. For example, anti-cancer drugs usedin cancers that lead to bone lesions will often need to beco-administered with compounds for use in the prevention or treatment ofbone-related disorders or conditions. Many anti-cancer drugs have anarrow therapeutic window (i.e. small margin between efficacious andtoxic dose) and it is therefore critically important that plasma levelsof such drugs are carefully controlled in order to avoid toxicity orreduced efficacy. One example of such an anti-cancer drug that is usedas a primary treatment for multiple myeloma is the proteasome inhibitorbortezomib (known as Velcade), which has a very narrow therapeuticmargin. This drug is extensively metabolised by CYP3A4 such thatpatients receiving co-administration of any compound that may induce orinhibit this enzyme require close monitoring (see Highlights ofPrescribing Information for Velcade:http://www.accessdata.fda.gov/drugsatfda_docs/label/2008/021602s0151b1.pdf).Accordingly, there is a need for novel compounds for use in theprevention or treatment of bone-related disorders or conditions thatdisplay low activity against liver enzymes such as CYP3A4. A particularcompound of the invention, namely3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidetri-hydrochloride, has now surprisingly been found to possess such lowactivity against CYP3A4. A further advantageous property of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidetri-hydrochloride, is that it does not display potent inhibitoryactivity against the cardiac Na⁺ channel, hNav1.5.

Furthermore, certain compounds of the invention also display usefulselectivity against GSK3 when compared to other particular kinases.

BRIEF DESCRIPTION OF FIGURES

FIG. 1A: Images showing Alizarin red S staining of mineralised bonenodules in response to 300 nM3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidetri-hydrochloride compared with vehicle (0.2% DMSO) after 24 days ofhuman adipose-derived stem cell culture. Experiments were performed intriplicate.

FIG. 1B: Effect of 300 nM3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidetri-hydrochloride on bone mineralization, Alizarin red S stainingquantified using cetylpyridinium chloride extraction. Data are shown asmean absorbance at 550 nm and analysed using one-way ANOVA followed byTukey's multiple comparison post hoc test: n=9; ***, P<0.001.

FIG. 2: Serum P1NP levels following 7 days of oral dosing with3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidetri-hydrochloride in rats. (Results expressed as mean±sem and *p<0.05,when a statistically significant difference was observed betweencompound treatment and vehicle control group.

FIG. 3: Serum TRAcP-5b levels following 7 days of oral dosing with3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidetri-hydrochloride in rats. (Results expressed as mean±sem and *p<0.05 or**p<0.01, when a statistically significant difference was observedbetween compound treatment and vehicle control group).

FIG. 4: X-Ray Powder Diffraction Pattern of Form B of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamide.

FIG. 5: DSC Thermogram of Form B of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamide.

FIG. 6 a: X-Ray Powder Diffraction Pattern for Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate.

FIG. 6 b: X-Ray Powder Diffraction Pattern for Form C of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate.

FIG. 7 a: DSC-Thermogram for Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate.

FIG. 7 b: DSC-Thermogram for Form C of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate.

FIG. 8: X-Ray Powder Diffraction Pattern Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-tosylate.

FIG. 9: DSC-Thermogram for Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-tosylate.

FIG. 10: X-Ray Powder Diffraction Pattern for Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidedi-esylate.

FIG. 11: DSC-Thermogram for Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidedi-esylate.

FIG. 12: X-Ray Powder Diffraction Pattern for Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-DL-mandelate.

FIG. 13: DSC-Thermogram for Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-DL-mandelate.

FIG. 14: X-Ray Powder Diffraction Pattern for Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-benzoate.

FIG. 15: DSC-Thermogram for Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-benzoate.

According to one aspect of the invention there is provided a pyrazinederivative of the Formula I

in which:n is 2 or 3;m is 2 or 3;R is methyl or ethyl; or a pharmaceutically-acceptable salt thereof.

According to a further aspect of the invention there is provided apyrazine derivative of the Formula I, which is3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamide:

or a pharmaceutically-acceptable salt thereof.

It is to be understood that compounds of Formula I defined above mayexhibit the phenomenon of tautomerism. It is to be understood that thepresent invention includes in its definition any such tautomeric form,or mixture of different tautomeric forms, which possess GSK3 enzymeinhibitory activity and is not to be limited merely to any onetautomeric form utilised within the formulae drawings or named in theExamples. In general, just one of any such tautomeric forms is named inthe Examples that follow hereinafter or is presented in any relevantformulae drawings that follow hereinafter.

A suitable pharmaceutically-acceptable salt of a compound of the FormulaI is, for example, an acid-addition salt of a compound of the Formula I,for example an acid-addition salt with an inorganic or organic acid suchas hydrochloric, hydrobromic, sulphuric, trifluoroacetic or citric acid.A further suitable pharmaceutically-acceptable salt of a compound of theFormula I is, for example, a salt formed within the human or animal bodyafter administration of a compound of the Formula I.

A particular pharmaceutically-acceptable salt of a compound of theFormula I is, an acid-addition salt of a compound of the Formula I withan acid selected from hydrochloric, hydrobromic, sulphuric,trifluoroacetic, citric, benzenesulphonic, p-toluenesulphonic,ethanesulphonic, DL-mandelic or benzoic acid.

According to one aspect of the invention, a particularpharmaceutically-acceptable salt of the compound of the Formula I is anacid-addition salt of a compound of the Formula I with hydrochloricacid.

According to a further aspect of the invention, a particularpharmaceutically-acceptable salt of the compound of the Formula I is anacid-addition salt of a compound of the Formula I with benzenesulphonicor p-toluenesulphonic acid.

According to a further aspect of the invention, a particularpharmaceutically-acceptable salt of the compound of the Formula I is anacid-addition salt of a compound of the Formula I with benzenesulphonicacid.

According to a further aspect of the invention, a particularpharmaceutically-acceptable salt of the compound of the Formula I is anacid-addition salt of a compound of the Formula I withp-toluenesulphonic acid.

According to a further aspect of the invention, a particularpharmaceutically-acceptable salt of the compound of the Formula I is anacid-addition salt of a compound of the Formula I with an acid selectedfrom ethanesulphonic, DL-mandelic or benzoic acid.

It is further to be understood that a suitablepharmaceutically-acceptable solvate of a compound of the Formula I alsoforms an aspect of the present invention. A suitablepharmaceutically-acceptable solvate is, for example, a hydrate such as ahemi-hydrate, a mono-hydrate, a di-hydrate or a tri-hydrate or analternative quantity thereof.

Some compounds of the Formula I may exhibit polymorphism. It is to beunderstood that the present invention encompasses any polymorphic form,or mixtures thereof, which form possesses properties useful in theinhibition of glycogen synthase kinase-3 activity, it being well knownin the art how to determine efficacy of a polymorphic form for theinhibition of glycogen synthase kinase-3 activity by the standard testsdescribed hereinafter.

It is generally known that crystalline materials may be analysed usingconventional techniques such as X-Ray Powder Diffraction (hereinafterXRPD) analysis, Differential Scanning Calorimetry (hereinafter DSC),Thermal Gravimetric Analysis (hereinafter TGA), Diffuse ReflectanceInfrared Fourier Transform (DRIFT) spectroscopy, Near Infrared (NIR)spectroscopy, solution and/or solid state nuclear magnetic resonancespectroscopy. The water content of such crystalline materials may bedetermined by Karl Fischer analysis.

As an example, a particular crystalline form has been identified for thecompound of Example 2.

Accordingly, a further aspect of the invention is Form B of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamide.

All of the following XRPD data was measured as described in paragraph(viii) in the introduction of the Examples section.

According to a further aspect of the present invention, there isprovided a crystalline form, Form B of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamide,which has an X-ray powder diffraction pattern with at least one specificpeak at 2-theta=3.5° plus or minus 0.1° 2-theta.

According to a further aspect of the present invention, there isprovided a crystalline form, Form B of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamide,which has an X-ray powder diffraction pattern with at least one specificpeak at 2-theta=17.6° plus or minus 0.1° 2-theta.

According to a further aspect of the present invention, there isprovided a crystalline form, Form B of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamide,which has an X-ray powder diffraction pattern with at least two specificpeaks at 2-theta=3.5° and 17.6° wherein said values may be plus or minus0.1° 2-theta.

According to a further aspect of the present invention, there isprovided a crystalline form, Form B of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamide,which has an X-ray powder diffraction pattern with specific peaks at2-theta=3.5, 7.0, 9.5, 10.4, 12.4, 13.8, 14.1, 15.9, 17.6, 21.0° whereinsaid values may be plus or minus 0.1° 2-theta.

According to a further aspect of the present invention, there isprovided a crystalline form, Form B of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamide,which has an X-ray powder diffraction pattern substantially the same asthe X-ray powder diffraction pattern shown in FIG. 4.

According to a further aspect of the present invention, there isprovided a crystalline form, Form B of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamide,which has an X-ray powder diffraction pattern with at least one specificpeak at 2-theta=3.5°.

According to a further aspect of the present invention, there isprovided a crystalline form, Form B of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamide,which has an X-ray powder diffraction pattern with at least one specificpeak at 2-theta=17.6°.

According to a further aspect of the present invention, there isprovided a crystalline form, Form B of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamide,which has an X-ray powder diffraction pattern with at least two specificpeaks at 2-theta=3.5° and 17.6°.

According to a further aspect of the present invention, there isprovided a crystalline form, Form B of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamide,which has an X-ray powder diffraction pattern with specific peaks at2-theta=3.5, 7.0, 9.5, 10.4, 12.4, 13.8, 14.1, 15.9, 17.6, 21.0°.

According to a further aspect of the present invention, there isprovided a crystalline form, Form B of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamide,which has an X-ray powder diffraction pattern as shown in FIG. 4.

As a further example, the compound of Example 3 exhibits polymorphismand two crystalline forms have been identified.

Accordingly, a further aspect of the invention is Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate, which has an X-ray powder diffraction pattern with atleast one specific peak at about 2-theta=8.4°.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate, which has an X-ray powder diffraction pattern with atleast one specific peak at about 2-theta=14.5°.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate, which has an X-ray powder diffraction pattern with atleast two specific peaks at about 2-theta=8.4° and 14.5°.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate, which has an X-ray powder diffraction pattern withspecific peaks at about 2-theta=7.5, 8.4, 11.0, 14.5, 15.7, 18.5, 20.2,22.0, 22.2, 23.0°.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate, which has an X-ray powder diffraction patternsubstantially the same as the X-ray powder diffraction pattern shown inFIG. 6 a.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate, which has an X-ray powder diffraction pattern with atleast one specific peak at 2-theta=8.4° plus or minus 0.1° 2-theta.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate, which has an X-ray powder diffraction pattern with atleast one specific peak at 2-theta=14.5° plus or minus 0.1° 2-theta.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate, which has an X-ray powder diffraction pattern with atleast two specific peaks at 2-theta=8.4° and 14.5° wherein said valuesmay be plus or minus 0.1° 2-theta.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate, which has an X-ray powder diffraction pattern withspecific peaks at 2-theta=7.5, 8.4, 11.0, 14.5, 15.7, 18.5, 20.2, 22.0,22.2, 23.0° wherein said values may be plus or minus 0.1° 2-theta.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate, which has an X-ray powder diffraction pattern with atleast one specific peak at 2-theta=8.4°.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate, which has an X-ray powder diffraction pattern with atleast one specific peak at 2-theta=14.5°.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate, which has an X-ray powder diffraction pattern with atleast two specific peaks at 2-theta=8.4° and 14.5°.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate, which has an X-ray powder diffraction pattern withspecific peaks at 2-theta=7.5, 8.4, 11.0, 14.5, 15.7, 18.5, 20.2, 22.0,22.2, 23.0°.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate, which has an X-ray powder diffraction pattern as shown inFIG. 6 a.

Accordingly, a further aspect of the invention is Form C of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate.

According to a further aspect of the present invention, there isprovided a crystalline form, Form C of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate, which has an X-ray powder diffraction pattern with atleast two specific peaks at about 2-theta=11.2°, 14.7° and 15.8°.

According to a further aspect of the present invention, there isprovided a crystalline form, Form C of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate, which has an X-ray powder diffraction pattern withspecific peaks at about 2-theta=11.2, 12.6, 14.7, 15.5, 15.8, 16.8,20.4, 21.1, 22.5, 26.5°.

According to a further aspect of the present invention, there isprovided a crystalline form, Form C of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate, which has an X-ray powder diffraction patternsubstantially the same as the X-ray powder diffraction pattern shown inFIG. 6 b.

According to a further aspect of the present invention, there isprovided a crystalline form, Form C of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate, which has an X-ray powder diffraction pattern with atleast one specific peak at 2-theta=11.2° plus or minus 0.1° 2-theta.

According to a further aspect of the present invention, there isprovided a crystalline form, Form C of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate, which has an X-ray powder diffraction pattern with atleast one specific peak at 2-theta=14.7° plus or minus 0.1° 2-theta.

According to a further aspect of the present invention, there isprovided a crystalline form, Form C of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate, which has an X-ray powder diffraction pattern with atleast one specific peak at 2-theta=15.8° plus or minus 0.1° 2-theta.

According to a further aspect of the present invention, there isprovided a crystalline form, Form C of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate, which has an X-ray powder diffraction pattern with atleast two specific peaks at 2-theta=11.2°, 14.7° and 15.8° wherein saidvalues may be plus or minus 0.1° 2-theta.

According to a further aspect of the present invention, there isprovided a crystalline form, Form C of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate, which has an X-ray powder diffraction pattern withspecific peaks at 2-theta=11.2, 12.6, 14.7, 15.5, 15.8, 16.8, 20.4,21.1, 22.5, 26.5°, wherein said values may be plus or minus 0.1°2-theta.

According to a further aspect of the present invention, there isprovided a crystalline form, Form C of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate, which has an X-ray powder diffraction pattern as shown inFIG. 6 b.

As yet a further example, a particular crystalline form has beenidentified for the compound of Example 4.1.

Accordingly, a further aspect of the invention is Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-tosylate.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-tosylate, which has an X-ray powder diffraction pattern with atleast one specific peak at about 2-theta=8.0°.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-tosylate, which has an X-ray powder diffraction pattern with atleast one specific peak at about 2-theta=23.0°.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-tosylate, which has an X-ray powder diffraction pattern with atleast two specific peaks at about 2-theta=8.0° and 23.0°.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-tosylate, which has an X-ray powder diffraction pattern withspecific peaks at about 2-theta=8.0, 11.7, 14.8, 20.0, 20.9, 21.5, 22.2,23.0, 27.5, 28.0°.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-tosylate, which has an X-ray powder diffraction patternsubstantially the same as the X-ray powder diffraction pattern shown inFIG. 8.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-tosylate, which has an X-ray powder diffraction pattern with atleast one specific peak at 2-theta=8.0° plus or minus 0.1° 2-theta.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-tosylate, which has an X-ray powder diffraction pattern with atleast one specific peak at 2-theta=23.0° plus or minus 0.1° 2-theta.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-tosylate, which has an X-ray powder diffraction pattern with atleast two specific peaks at 2-theta=8.0° and 23.0° wherein said valuesmay be plus or minus 0.1° 2-theta.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-tosylate, which has an X-ray powder diffraction pattern withspecific peaks at 2-theta=8.0, 10.5, 11.7, 12.4, 14.8, 16.0, 17.0, 18.1,20.0, 20.9, 21.5, 22.2, 23.0, 27.5, 28.0° wherein said values may beplus or minus 0.1° 2-theta.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-tosylate, which has an X-ray powder diffraction pattern with atleast one specific peak at 2-theta=8.0°.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-tosylate, which has an X-ray powder diffraction pattern with atleast one specific peak at 2-theta=23.0°.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-tosylate, which has an X-ray powder diffraction pattern with atleast two specific peaks at 2-theta=8.0° and 23.0°.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-tosylate, which has an X-ray powder diffraction pattern withspecific peaks at 2-theta=8.0, 10.5, 11.7, 12.4, 14.8, 16.0, 17.0, 18.1,20.0, 20.9, 21.5, 22.2, 23.0, 27.5, 28.0°.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-tosylate, which has an X-ray powder diffraction pattern as shown inFIG. 8.

Further examples of compounds for which particular crystalline formshave been identified are the compounds of Examples 4.2-4.4.

Accordingly, a further aspect of the invention is Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidedi-esylate.

Accordingly, a further aspect of the invention is Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-DL-mandelate.

Accordingly, a further aspect of the invention is Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-benzoate.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidedi-esylate, which has an X-ray powder diffraction pattern with at leasttwo specific peaks at 2-theta=5.2° and 12.7° wherein said values may beplus or minus 0.1° 2-theta.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidedi-esylate, which has an X-ray powder diffraction pattern with specificpeaks at 2-theta=5.2, 10.4, 12.7, 15.0, 16.9, 17.3, 19.1, 19.6, 20.0,23.5° wherein said values may be plus or minus 0.1° 2-theta.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidedi-esylate, which has an X-ray powder diffraction pattern as shown inFIG. 10.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-DL-mandelate, which has an X-ray powder diffraction pattern with atleast two specific peaks at 2-theta=2.7° and 7.8° wherein said valuesmay be plus or minus 0.1° 2-theta.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-DL-mandelate, which has an X-ray powder diffraction pattern withspecific peaks at 2-theta=2.7, 5.2, 7.8, 9.6, 10.5, 12.3, 13.1, 15.7,18.4° wherein said values may be plus or minus 0.1° 2-theta.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-DL-mandelate, which has an X-ray powder diffraction pattern asshown in FIG. 12.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-benzoate, which has an X-ray powder diffraction pattern with atleast two specific peaks at 2-theta=5.3° and 9.2° wherein said valuesmay be plus or minus 0.1° 2-theta.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-benzoate, which has an X-ray powder diffraction pattern withspecific peaks at 2-theta=5.3, 6.2, 9.2, 9.8, 11.9, 16.6, 17.8, 18.1,19.5, 24.6° wherein said values may be plus or minus 0.1° 2-theta.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-benzoate, which has an X-ray powder diffraction pattern as shown inFIG. 14.

It will be understood that 2-theta values of the X-ray powderdiffraction patterns may vary slightly from one machine to another orfrom one sample to another, and so the values quoted are not to beconstrued as absolute.

It is also known that an X-ray powder diffraction pattern may beobtained which has one or more measurement errors depending onmeasurement conditions (such as equipment or machine used). Inparticular, it is generally known that intensities in an X-ray powderdiffraction pattern may fluctuate depending on measurement conditions.Therefore it should be understood that the crystalline Forms of thepresent invention described above, unless otherwise stated, are notlimited to the crystals that provide X-ray powder diffraction patternsidentical to the X-ray powder diffraction pattern shown in FIGS. 4, 6a,6b, 8, 10, 12 and 14, and any crystals providing X-ray powderdiffraction patterns substantially the same as those shown in theseFigures fall within the scope of the present invention. A person skilledin the art of X-ray powder diffraction is able to judge the substantialidentity of X-ray powder diffraction patterns.

Persons skilled in the art of X-ray powder diffraction will also realisethat the relative intensity of peaks can be affected by, for example,grains above 30 microns in size and non-unitary aspect ratios, which mayaffect analysis of samples. The skilled person will also realise thatthe position of reflections can be affected by the precise height atwhich the sample sits in the diffractometer and the zero calibration ofthe diffractometer. The surface planarity of the sample may also have asmall effect. Hence the diffraction pattern data presented are not to betaken as absolute values (see Jenkins, R & Snyder, R. L. ‘Introductionto X-Ray Powder Diffractometry’ John Wiley & Sons 1996; Bunn, C. W.(1948), Chemical Crystallography, Clarendon Press, London; Klug, H. P. &Alexander, L. E. (1974), X-Ray Diffraction Procedures).

Generally, a measurement error of a diffraction angle in an X-ray powderdiffractogram is approximately plus or minus 0.1° 2-theta, and suchdegree of a measurement error should be taken into account whenconsidering the X-ray powder diffraction data. Furthermore, it should beunderstood that intensities might fluctuate depending on experimentalconditions and sample preparation (preferred orientation).

It is further to be understood that a suitablepharmaceutically-acceptable pro-drug of a compound of the Formula I alsoforms an aspect of the present invention. Accordingly, the compound ofthe invention may be administered in the form of a pro-drug, that is acompound that is broken down in the human or animal body to release acompound of the invention. A pro-drug may be used to alter the physicalproperties and/or the pharmacokinetic properties of a compound of theinvention. A pro-drug can be formed when the compound of the inventioncontains a suitable group or substituent to which a property-modifyinggroup can be attached. Examples of pro-drugs include in vivo cleavableamide derivatives that may be formed at an amino group in a compound ofthe Formula I.

Accordingly, the present invention includes a compound of the Formula Ias defined hereinbefore when made available by organic synthesis andwhen made available within the human or animal body by way of cleavageof a pro-drug thereof. Accordingly, the present invention includes thosecompounds of the Formula I that are produced by organic synthetic meansand also such compounds that are produced in the human or animal body byway of metabolism of a precursor compound, that is a compound of theFormula I may be a synthetically-produced compound or ametabolically-produced compound.

A suitable pharmaceutically-acceptable pro-drug of a compound of theFormula I is one that is based on reasonable medical judgement as beingsuitable for administration to the human or animal body withoutundesirable pharmacological activities and without undue toxicity.

It is to be understood that, insofar as a pharmaceutically-acceptablepro-drug of a compound of the Formula I defined above may exist inoptically active or racemic forms by virtue of one or more asymmetriccarbon atoms, the invention includes in its definition any suchoptically active or racemic form. The synthesis of optically activeforms may be carried out by standard techniques of organic chemistrywell known in the art, for example by synthesis from optically activestarting materials or by resolution of a racemic form.

Various forms of pro-drug have been described, for example in thefollowing documents:—

-   a) Methods in Enzymology, Vol. 42, p. 309-396, edited by K. Widder,    et al. (Academic Press, 1985);-   b) Design of Pro-drugs, edited by H. Bundgaard, (Elsevier, 1985);-   c) A Textbook of Drug Design and Development, edited by    Krogsgaard-Larsen and H. Bundgaard, Chapter 5 “Design and    Application of Pro-drugs”, by H. Bundgaard p. 113-191 (1991);-   d) H. Bundgaard, Advanced Drug Delivery Reviews, 8, 1-38 (1992);-   e) H. Bundgaard, et al., Journal of Pharmaceutical Sciences, 77, 285    (1988);-   f) N. Kakeya, et al., Chem. Pharm. Bull., 32, 692 (1984);-   g) T. Higuchi and V. Stella, “Pro-Drugs as Novel Delivery Systems”,    A.C.S. Symposium Series, Volume 14; and-   h) E. Roche (editor), “Bioreversible Carriers in Drug Design”,    Pergamon Press, 1987.

A suitable pharmaceutically-acceptable pro-drug of the compound of theFormula I that possesses an amino group is, for example, an in vivocleavable amide derivative thereof. Suitable pharmaceutically-acceptableamides from an amino group include, for example an amide formed with(1-10C)alkanoyl groups such as an acetyl, benzoyl, phenylacetyl andsubstituted benzoyl and phenylacetyl groups. Examples of ringsubstituents on the phenylacetyl and benzoyl groups include aminomethyl,N-alkylaminomethyl, N,N-dialkylaminomethyl, morpholinomethyl,piperazin-1-ylmethyl and 4-(1-4C)alkylpiperazin-1-ylmethyl.

The in vivo effects of a compound of the Formula I may be exerted inpart by one or more metabolites that are formed within the human oranimal body after administration of a compound of the Formula I. Asstated hereinbefore, the in vivo effects of a compound of the Formula Imay also be exerted by way of metabolism of a precursor compound (apro-drug).

Particular compounds of the invention are each of the Examples andpharmaceutically-acceptable salt(s) thereof, each of which provides afurther independent aspect of the invention.

According to a further aspect of the invention there is provided apyrazine derivative of the Formula I or a pharmaceutically-acceptablesalt thereof, which is obtainable by following any of the Examples asdisclosed herein.

A further feature of the invention is any of the scopes defined hereinwith the proviso that specific Examples, such as Example 1, 2, 3a, 3b,4.1 etc. are individually disclaimed.

A yet further feature of the invention is any of the scopes definedherein with the proviso that one specific Example, such as Example 1, 2,3a, 3b, 4.1 etc. is individually disclaimed.

Particular novel compounds of the invention include, for example,pyrazine derivatives of the Formula I, or pharmaceutically-acceptablesalts thereof, wherein, each of n, m and R has any of the meaningsdefined hereinbefore or in paragraphs (a) to (h) hereinafter:—

(a) n is 2;(b) n is 3;(c) m is 2;(d) m is 3;(e) n is 3 and m is 3;(f) n is 3 and m is 2;(g) n is 2 and m is 3; or(h) R is methyl.

A particular group of compounds of the invention are pyrazinederivatives of Formula I above wherein:—

n is 2 or 3;m is 3;R is methyl or ethyl, conveniently methyl; or apharmaceutically-acceptable salt thereof.

A particular group of compounds of the invention are pyrazinederivatives of Formula I above wherein:—

n is 3;m is 2 or 3;R is methyl or ethyl, conveniently methyl; or apharmaceutically-acceptable salt thereof.

A particular compound of the invention is a pyrazine derivative of theFormula I selected from any one of the following:—

-   3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamide;-   3-amino-6-(4-{2-[(2-methoxyethyl)amino]ethyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamide;-   3-amino-6-(4-{2-[(3-methoxypropyl)amino]ethyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamide;    and-   3-amino-6-(4-{3-[(2-methoxyethyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamide;    or a pharmaceutically-acceptable salt thereof.

According to a yet further aspect of the invention, a particularcompound of the invention is a pyrazine derivative of the Formula Iselected from any one of the following:—

-   3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamide;-   3-amino-6-(4-{2-[(3-methoxypropyl)amino]ethyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamide;    and-   3-amino-6-(4-{3-[(2-methoxyethyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamide;    or a pharmaceutically-acceptable salt thereof.

Another aspect of the present invention provides a process for preparinga compound of the Formula I, or a pharmaceutically-acceptable saltthereof. A suitable process is illustrated by the followingrepresentative process variants in which, unless otherwise stated, n, mand R have any of the meanings defined hereinbefore. Necessary startingmaterials may be obtained by standard procedures of organic chemistry.The preparation of such starting materials is described in conjunctionwith the following representative process variants and within theaccompanying Examples. Alternatively necessary starting materials areobtainable by analogous procedures to those illustrated, which arewithin the ordinary skill of an organic chemist.

Suitable process variants include, for example, the following:—

(a) An amine displacement reaction of a compound of the Formula II:

or a reactive derivative thereof, wherein m is 2 or 3 and X represents asuitable leaving group such as for example a halogen (such as bromo,fluoro, chloro or iodo), or a sulphonate group such as for examplemethane sulphonate or trifluoromethane sulphonate, wherein anyfunctional group present is protected if necessary, with an aminecompound of the Formula III:

wherein n is 2 or 3 and R is methyl or ethyl, to form a compound of theFormula I, whereafter any protecting group that is present is removed.

The reaction is conveniently carried out in the presence of an organicsolvent such as dichloromethane, N,N-dimethylformamide, dimethylsulphoxide or acetonitrile in the presense of a suitable base such asfor example an alkali metal carbonate (such as for example sodiumcarbonate or potassium carbonate) or an alkaline earth metal carbonate(such as for example calcium carbonate), a metal hydroxide (such as forexample sodium hydroxide or potassium hydroxide) at a temperature, forexample, in the range from 0 to 150° C., conveniently at a temperaturerange of between 80 and 90° C.

Compounds of the Formula II, including reactive derivatives may, forexample, be prepared by the reaction of a compound of the Formula IV:

wherein m is 2 or 3, with a suitable activating reagent such as forexample, methanesulphonyl chloride, toluensulphonyl chloride, thionylchloride or oxalyl chloride. The reaction may conveniently be carriedout in an organic solvent such as tetrahydrofuran or dichloromethaneusing either an inorganic base such as sodium carbonate or potassiumcarbonate, or an organic base such as triethyl amine ordiisopropylethylamine at a temperature in the range from 0 to 150° C.,conveniently at ambient temperature.

Compounds of the Formula IV, may for example, be prepared by thereaction of a compound of the Formula V:

wherein m is 2 or 3 and PG represents a protecting group such astert-butyldimethylsilyl or tetrahydropyrane with a suitable acid such asfor example sulphuric acid.

The reaction may conveniently be carried out in an organic solvent suchas methanol, tetrahydrofuran or dioxane using either an inorganic acidsuch as hydrochloric acid or hydrobromic acid, or an organic acid suchas trifluoroacetic acid at a temperature, for example, in the range from0 to 150° C., conveniently at a temperature range of between 20 and 80°C.

Compounds of the Formula V may, for example, be prepared by a metalcatalysed coupling reaction of a compound of the Formula VI:

wherein Y represents a suitable leaving group such as for example ahalogen (such as chloro, fluoro, bromo or iodo), ortrifluoromethanesulphonate, with a compound of the Formula VII:

to wherein m is 2 or 3, Z represents a suitable leaving group such asfor example a halogen (such as chloro, fluoro, bromo or iodo) ortrifluoromethanesulphonate and PG represents a protecting group such astert-butyldimethylsilyl or tetrahydropyrane.

The reaction may conveniently be carried out in an organic solvent suchas tetrahydrofuran, N,N-dimethylformamide, or dioxane in the presense ofa Pd catalyst with a suitable ligand such as Pd(dppf)Cl₂ or Pd(PPh₃)₃and in the presence of a diborane with a suitable ligand such asbis(pinacolato)diboron or bis(catecholato)diboron and in a presence of asalt such as potassium acetate or sodium acetate at a temperature in therange from 0 to 150° C., conveniently at a temperature range between 80and 90° C.

Alternatively, compounds of the Formula IV, may for example, be preparedby the reaction of a compound of the Formula VI with a compound of theFormula VIII:

wherein m is 2 or 3 and W represents a boronic acid or boronic acidester such as for example pinacole boronate or catechol boronate.

The reaction may conveniently be carried out in an organic solvent suchas tetrahydrofuran, N,N-dimethylformamide, or dioxane in the presense ofa Pd catalyst with a suitable ligand such as Pd(dppf)Cl₂ or Pd(PPh₃)₃and in the presence of a salt such as potassium acetate or sodiumacetate at a temperature in the range from 0 to 150° C., conveniently ata temperature range between 70 and 90° C.

Compounds of the Formula IV could also be prepared using an inversecoupling process to that described above, wherein the compound ofFormula VI would have a W group (which is a a boronic acid or boronicacid ester such as for example pinacole boronate or catechol boronate asdefined hereinbefore) in place of the Y group and the compound ofFormula VIII would have a Y group (being a suitable leaving group suchas for example a halogen (such as chloro, fluoro, bromo or iodo), ortrifluoromethanesulphonate as defined hereinbefore) present in place ofthe W group. Suitable conditions for the reaction would be thosedescribed above.

Compounds of the Formula VIII may, for example, be prepared by treatmentof a compound of the Formula IX:

wherein m is 2 or 3 and Z represents a suitable leaving group such asfor example a halogen or trifluoromethane sulphonate, with a suitablediborane complex such as for example, bis(pinacolato)diboron orbis(catecholato)diboron. The reaction may conveniently be carried out inan organic solvent such as tetrahydrofuran, N,N-dimethylformamide,acetonitrile or dioxane in the presense of a Pd catalyst such as Pd—Cl₂or Pd(OAc)₂ with a ligand such as PCy₃ or PPh₃ and in the presence of asalt such as potassium acetate or sodium acetate at a temperature in therange from 0 to 150° C., conveniently at a temperature range between 70and 90° C.

Compounds of the Formula VII, may, for example, be prepared by treatmentof a compound of the Formula IX:

wherein m is 2 or 3 and Z represents a suitable leaving group such asfor example a halogen or trifluoromethane sulphonate, with a suitableprotecting agent such as for example tert-butyldimethylsilyl chloride.The reaction is conveniently conducted in the presence of an organicsolvent such as tetrahydrofuran or dichloromethane using either aninorganic base such as sodium carbonate or potassium carbonate, or anorganic base such as triethyl amine or diisopropylethylamine and in thepresence of a suitable catalyst such as 4-dimethylaminopyridine at atemperature in the range from 0 to 150° C., conveniently at ambienttemperature.

Alternatively, the above reaction can be conducted with dihydropyran inan organic solvent such as THF or if no solvent is presents with acatalyst such as sulphuric acid or HCl at a temperature in the rangefrom 0 to 150° C., conveniently at refluxing conditions.

Compounds of the Formulae III, VI and IX may be obtained by conventionalprocedures or are commercially available, known in the literature, orthey can be prepared by using or adapting standard processes known inthe art;

(b) A reaction of a compound of the Formula X:

wherein, n is 2 or 3, m is 2 or 3, R is methyl or ethyl and PG₂represents a suitable protecting group such as for example analkoxylcarbonyl (such as t-butoxycarbonyl or isopropoxycarbonyl), with asuitable acid such as for example HCl or trifluoroacetic acid.

The reaction may conveniently carried out in an organic solvent such asEtOAc, methanol, 2-methyltetrahydrofuran or tetrahydrofuran, or aninorganic solvent such as for example water using either an inorganicacid such as hydrochloric acid or hydrobromic acid, or an organic acidsuch as trifluoroacetic acid at a temperature, for example, in the rangefrom 0 to 150° C., conveniently at a temperature range of between 20 and80° C.

Compounds of the Formula X may, for example, be prepared by a metalcatalysed coupling reaction of a compound of the Formula XI:

wherein, n is 2 or 3, m is 2 or 3, R is methyl or ethyl, PG₂ representsa suitable protecting group such as for example an alkoxylcarbonyl (suchas t-butoxycarbonyl or isopropoxycarbonyl), and W represents a boronicacid or a boronic acid ester such as for example pinacole boronate orcatechol boronate, with a compound of the Formula VI.

The reaction may conveniently be carried out in an organic solvent suchas tetrahydrofuran, N,N-dimethylformamide, dioxane or 1-butanol in thepresense of a Pd catalyst with a suitable ligand such as Pd(dppf)Cl₂ orPd(PPh₃)₃, or alternatively a mixture with sodiumtetrachloropalladateand 3-(di-tert-butylphosphonium)propane sulphate, and in the presence ofa salt such as potassium acetate, sodium acetate or potassium phosphatetribasic N-hydrate at a temperature in the range from 0 to 150° C.,conveniently at a temperature range between 70 and 90° C.

Compounds of the Formula XI may, for example, be prepared by thereaction of a compound of the Formula XII:

wherein, n is 2 or 3, m is 2 or 3, R is methyl or ethyl, PG₂ representsa suitable protecting group such as for example an alkoxylcarbonyl (suchas t-butoxycarbonyl or isopropoxycarbonyl) and Z represents a suitableleaving group such as for example a halogen or trifluoromethanesulphonate with a suitable diborane complex such as for example,bis(pinacolato)diboron or bis(catecholato)diboron. The reaction mayconveniently be carried out in an organic solvent such astetrahydrofuran, N,N-dimethylformamide, acetonitrile or dioxane in thepresense of a Pd catalyst such as Pd—Cl₂ or Pd(OAc)₂ with a ligand suchas PCy₃ or PPh₃ and in the presence of a salt such as potassium acetateor sodium acetate at a temperature in the range from 0 to 150° C.,conveniently at a temperature range between 70 and 90° C.

Compounds of the Formula XII may, for example, be prepared by thereaction of a compound of the Formula XIII:

wherein, n is 2 or 3, m is 2 or 3, R is methyl or ethyl, and Zrepresents a suitable leaving group such as for example a halogen ortrifluoromethane sulphonate with a suitable protecting agent such as forexample Di-tert-butyl dicarbonate. The reaction may conveniently becarried out in an organic solvent such as methanol, tetrahydrofuran ordioxane using either an inorganic acid such as hydrochloric acid orhydrobromic acid, or an organic acid such as trifluoroacetic acid at atemperature, for example, in the range from 0 to 150° C., convenientlyat a temperature range of between 20 and 80° C.

Compounds of the Formula XIII may, for example, be prepared by thereaction of a compound of the Formula XIV:

or a reactive derivative thereof, wherein m is 2 or 3, X represents asuitable leaving group such as for example a halogen (such as bromo,fluoro, chloro or iodo), or a sulphonate group such as for examplemethane sulphonate or trifluoromethane sulphonate and Z represents asuitable leaving group such as for example a halogen or trifluoromethanesulphonate with a suitable protecting agent such as for exampleDi-tert-butyl dicarbonate, with an amine compound of the Formula III.

The reaction may conveniently be carried out in the presence of anorganic solvent such as dichloromethane, N,N-dimethylformamide, dimethylsulphoxide or acetonitrile in the presense of a suitable base such asfor example an alkali metal carbonate (such as for example sodiumcarbonate or potassium carbonate) or an alkaline earth metal carbonate(such as for example calcium carbonate), a metal hydroxide (such as forexample sodium hydroxide or potassium hydroxide) at a temperature, forexample, in the range from 0 to 150° C., conveniently at a temperaturerange of between 80 and 90° C.

Compounds of the Formula XIV may, for example, be prepared by thereaction of a compound of the Formula IX, with a suitable activatingreagent such as for example, methanesulphonyl chloride, toluensulphonylchloride, thionyl chloride or oxalyl chloride. The reaction mayconveniently be carried out in an organic solvent such astetrahydrofuran or dichloromethane using either an inorganic base suchas sodium carbonate or potassium carbonate, or an organic base such astriethyl amine or diisopropylethylamine at a temperature in the rangefrom 0 to 150° C., conveniently at ambient temperature.

When a pharmaceutically-acceptable salt of a pyrazine derivative of theFormula I is required, for example an acid-addition salt, it may beobtained by, for example, reaction of said pyrazine derivative with asuitable acid.

When a pharmaceutically-acceptable pro-drug of a pyrazine derivative ofthe Formula I is required, it may be obtained using a conventionalprocedure. For example, an in vivo cleavable amide of a pyrazinederivative of the Formula I may be obtained by, for example, reaction ofa compound of the Formula I containing an amino group with apharmaceutically-acceptable carboxylic acid.

It will also be appreciated by the person skilled in the organicsynthetic arts that certain of the ring substituents in the compounds ofthe present invention may be introduced by standard aromaticsubstitution reactions or generated by conventional functional groupmodifications either prior to or immediately following the processesmentioned above, and as such are included in the process aspect of theinvention. Such reactions and modifications include, for example,introduction of a substituent by means of an aromatic substitutionreaction, reduction of substituents, alkylation of substituents,acylation of substituents, amidation of substituents and oxidation ofsubstituents. The reagents and reaction conditions for such proceduresare well known in the chemical art. Particular examples of aromaticsubstitution reactions include the introduction of an acyl group using,for example, an acyl halide and Lewis acid (such as aluminiumtrichloride) under Friedel Crafts conditions; the introduction of analkyl group using an alkyl halide and Lewis acid (such as aluminiumtrichloride) under Friedel Crafts conditions; and the introduction of ahalogeno group.

It will also be appreciated that, in some of the reactions mentionedhereinbefore, it may be necessary or desirable to protect any sensitivegroups in the compounds. The instances where protection is necessary ordesirable and suitable methods for protection are to known to thoseskilled in the art. Conventional protecting groups may be used inaccordance with standard practice (for illustration see T. W. Green,Protective Groups in Organic Synthesis, John Wiley and Sons, 1991).Thus, if reactants include groups such as amino, carboxy or hydroxy, itmay be desirable to protect the group in some of the reactions mentionedherein.

A suitable protecting group for an amino or alkylamino group is, forexample, an acyl group, for example an alkanoyl group such as acetyl, analkoxycarbonyl group, for example a methoxycarbonyl, ethoxycarbonyl ort-butoxycarbonyl group, an arylmethoxycarbonyl group, for examplebenzyloxycarbonyl, or an aroyl group, for example benzoyl. Thedeprotection conditions for the above protecting groups necessarily varywith the choice of protecting group. Thus, for example, an acyl groupsuch as an alkanoyl or alkoxycarbonyl group or an aroyl group may beremoved for example, by hydrolysis with a suitable base such as analkali metal hydroxide, for example lithium or sodium hydroxide.Alternatively an acyl group such as a t-butoxycarbonyl group may beremoved, for example, by treatment with a suitable acid as hydrochloric,sulphuric or phosphoric acid or trifluoroacetic acid and anarylmethoxycarbonyl group such as a benzyloxycarbonyl group may beremoved, for example, by hydrogenation over a catalyst such aspalladium-on-carbon, or by treatment with a Lewis acid for example borontris(trifluoroacetate). A suitable alternative protecting group for aprimary amino group is, for example, a phthaloyl group, which may beremoved by treatment with an alkylamine, for exampledimethylaminopropylamine, or with hydrazine.

The protecting groups may be removed at any convenient stage in thesynthesis using conventional techniques well known in the chemical art.

Certain of the intermediates defined herein are novel and these areprovided as a further feature of the invention. For example, compoundsof the Formula II and V (as defined hereinabove) may be useful asintermediates in the preparation of the compound of the invention.

Furthermore, the following compounds may be useful as intermediates inthe preparation of the compound of the invention:

-   3-amino-6-(4-(3-(tert-butyldimethylsilyloxy)propyl)phenyl)-N-(pyridin-3-yl)-pyrazine-2-carboxamide;    and-   3-(4-(5-Amino-6-(pyridin-3-ylcarbamoyl)pyrazin-2-yl)phenyl)propyl    methanesulfonate.

Biological Assays

The following assays can be used to measure the effects of the compoundsof the present invention:

In Vitro GSK3 Kinase Assay

This biochemical assay used a Fluorescence Resonance Energy Transfer(FRET)-based, coupled-enzyme format to determine the ability of testcompounds to inhibit substrate phosphorylation by recombinant GSK3βserine/threonine kinase (Kleman-Leyer, K. et al. Drug Disc. Devel. 2003,6:81-2). The substrate used in this assay was Z′-LYTE™ ser/Thr 9 peptideand the FRET assay kit was obtained from Invitrogen (Paisley, UK,catalogue number PV3324). N-terminal His6-tagged recombinant humanGSK30, with an H350L mutation, was expressed in baculovirus in Sf21insect cells, purified and obtained from Millipore (Billerica, Mass.,USA, catalogue number 14-306 Human).

Test compounds were prepared as 10 mM stock solutions indimethylsulphoxide (DMSO) and diluted in DMSO as required. Aliquots (2.5μl) of compound dilutions were added to the wells of an Optiplate-FPerkinElmer 384-well plate (Massachusetts, USA, catalogue number6007270). Control wells that produced a maximum signal corresponding tomaximum enzyme activity were created by using 100% phosphorylatedpeptide obtained from Invitrogen (Paisley, UK, catalogue number PV3326).A 5 μl aliquot of 100% phosphorylated peptide (100 μM in 1.33× kinasebuffer) was added to all maximum control wells. 2.5 μl of 4% DMSOsolution (v/v) in 1.33× kinase buffer was added to maximum controlwells. Control wells that produced a minimum signal corresponding to100% inhibited enzyme were created by adding 2.5 μl of 40 μMStaurosporine in 1.33× kinase buffer containing 4% DMSO (v/v). A 5 μlmixture of GSK3β (100 ng/μl) enzyme and non-phosphorylated substrate(Z-LYTE™ ser/Thr 9 peptide, 100 μM) in a 1.33× kinase buffer solutionwas added to each well except maximum control wells and the assay platecentrifuged at 270 g for 1 minute. The kinase buffer used 1.33× wasobtained from Invitrogen (Paisley, UK, catalogue number PV3189) andcontained 67 mM HEPES (pH 7.5), 13 mM MgCl₂, 1.3 mM EGTA, 0.01% BRIJ-35.Then 2.5 μl adenosine triphosphate (28 μM) in kinase buffer was addedper well, centrifuged at 270 g for 1 minute and then the assay plate wasshaken for 1 hour at room temperature. A proprietry development solutionwas prepared by adding 58 μl of development reagent A (Invitrogen,Paisley, UK, catalogue number PV3297) to 2142 μl of development reagentB (Invitrogen, Paisley, UK, catalogue number P3127). Then 5 μl ofInvitrogen propriety development solution was added per well,centrifuged at 270 g for 1 minute and then shaken for 1 hour at roomtemperature. The reaction was stopped by addition of 5 μl of a stopreagent per well (Invitrogen, Paisley, UK catalogue number P3094),centrifuged at 270 g for 1 minute and then shaken for 5 minutes at roomtemperature.

The resultant signals arising from laser light excitation at 400 nM wereread using a Perkin Elmer Envision spectrophotometer (Perkin Elmer,Massachusetts, USA). The Z′-LYTE™ biochemical assay employs aFRET-based, coupled-enzyme format and is based on the differentialsensitivity of phosphorylated and non-phosphorylated peptides toproteolytic cleavage. A ratiometric method, which calculates the ratio(the Emission Ratio) of donor emission to acceptor emission afterexcitation of the donor fluorophore at 400 nm, quantitates reactionprogress, as shown in the equation below.

Emission Ratio=Coumarin Emission (445 nm)÷Fluoresein Emission (520 nm)

The mean data values for each test compound concentration, 100% DMSOcontrol wells and 100% inhibition control wells were used to generate aconcentration response curve from which the percentage inhibition ofkinase activity with compound treatment was calculated and a potency(IC₅₀) value calculated. The IC₅₀ value is the concentration of testcompound that inhibited 50% of human GSK3 β kinase activity. The finalATP concentration in this assay was 7 μM.

(b) Cellular Beta-Catenin Stabilisation Assay

This assay uses an imaging based approach to measure the ability of testcompounds to promote Beta-catenin stabilisation in the nucleus ofC3H10T1/2 cells. Beta-catenin stabilisation is an indicator of GSK3βinhibition.

A C3H10T1/2 mouse clone 8 fibroblast cell line (American Type CultureCollection (ATCC, USA; catalogue number CRL 226) was routinelymaintained at 37° C. with 5% CO₂ in Basal Medium Eagle (BME, Invitrogen;Paisley, UK; catalogue number 41010) containing 10% (v/v) foetal bovineserum (FBS, Invitrogen, Paisley; UK; catalogue number 10270) and 2 mML-glutamine (Invitrogen, Paisley, UK; catalogue number 25030024). Forthe assay, the cells were detached from the culture flask with‘Accutase’ (Innovative Cell Technologies Inc., San Diego, USA; cataloguenumber AT104) using standard tissue culture methods and re-suspended inassay media comprising of BME containing 5% (v/v) FBS and 2 mML-glutamine to give 55,555 cells per ml. Then, 90 μl of this solutionwas seeded into each well of a clear-bottom 96 well tissue culture plate(Perkin Elmer Viewplate; catalogue number 6005182) using a MultidropCombi Dispenser (Thermo Fisher Scientific, Loughborough, UK) to give5,000 cells per well. Plates were incubated overnight at 37° C. with 5%CO₂ to allow the cells to adhere to the wells. Test compounds wereprepared as 10 mM stock solutions in dimethylsulphoxide (DMSO)(Sigma-Aldrich; Poole, UK, catalogue number 49442-9) and seriallydiluted in DMSO. A Multidrop Combi was used to dispense 245 μl ofpre-warmed assay media into a 96 well plate (Thermo Fisher Scientific,Loughborough, UK; catalogue number 50823925) before 5 μl of seriallydiluted compound was added to this intermediate plate. After mixing, 10μl of each compound concentration was added to the cell plates. Thetotal dilution factor from DMSO stock was 500 times. Control cellsreceived either DMSO (0.2% v/v final concentration) or 10 μM positivecontrol compound. The cells were incubated for 24 hours at 37° C. with5% CO₂.

After compound treatment the cells were fixed by the addition of 100 μlof 8% w/v paraformaldehyde (Sigma-Aldrich, Poole, UK; catalogue numberP6148), directly into the assay plate wells. The cells were incubated atambient temperature with fixative for 30 minutes before washing 3 timeswith 100 μl phosphate-buffered saline (PBS, Sigma-Aldrich; cataloguenumber D8537). The cells were then washed once and incubated at roomtemperature in blocking solution (PBS containing 1.1% w/v Bovine SerumAlbumin (BSA, Sigma-Aldrich, Poole, UK; catalogue number A7906) and 0.2%v/v Triton X-100 (Sigma-Aldrich, Poole, UK; catalogue number X100)) for1 hour. After removing the blocking solution, 30 μl of anti beta-cateninantibody (BD Biosciences; catalogue number 610154) at a concentration of1.25 μg/ml diluted in blocking solution, was added to the cells andincubated at 4° C. for 16 hours. The cells were then washed twice in 100μl blocking solution and incubated at ambient temperature for 1 hour.The cells were then placed in 40 μl of a secondary antibody solutioncomprising of AF647 donkey anti-mouse antibody (Invitrogen, Paisley, UK;catalogue number A31571) and Hoechst 33342 (Invitrogen, Paisley, UK;catalogue number H1399) nuclear counterstain at concentrations of 4μg/ml and 1 μM respectively diluted in blocking solution. Incubationwith the secondary staining solution was for 2 hours at roomtemperature. The cells were then washed 3 times in 100 μl of PBS beforethe plates were sealed ready for image capture. Fluorescent images werecaptured and quantified using the Incell Analyser 3000 (GE Healthcare,USA). Hoechst nuclear staining was visualised using an excitationwavelength of 365 nm along with a neutral density filter setting of 1and a 450-65 emission filter. Beta-catenin localisation was capturedusing a 633 nm laser wavelength, a neutral density filter of 1 and anemission filter of 695-55 nm. To enable a sufficient cell count, 4images per well were taken. Image data was analysed using the NuclearTrafficking Analysis Module, TRF2. Data from each well on an assay platewas normalised to the median values of the negative and positivecontrols from the same assay plate and percentage effect expressed as aproportion of the positive control. Data from wells where the total cellnumber fell below 50% of the experimental batch median was excluded fromanalysis. Concentration response curves were fitted to the followingform of the Hill equation (Neubig et al., 2003, Pharmacol Rev. 2003;55:597-606)

y=A+((B−A)÷(1+((C÷x)̂D)))

Where x=compound concentration (M) and y=% Effect. Also where A=basal ofconcentration effect curve, B=asymptote curve, C=midpoint of curve,D=slope Data for each test compound was reported as the compoundconcentration that results in half of the maximal effect (EC₅₀) and asthe maximum percentage effect.

The pharmacological properties of the compound of the Formula I may bedemonstrated at the following concentrations or doses in one or more ofthe above tests (a) and (b):

-   -   Test (a):—IC₅₀ versus human GSK3β enzyme in the range, for        example, 10 nM-1 μM    -   Test (b):—EC₅₀ versus cellular β-catenin accumulation in murine        C3H/10T1/2 cells, in the range, for example, 50 nM-5 μM.

For example, the pyrazine compound disclosed as Example 1 possessesactivity in Test (a) with an IC₅₀ versus human GSK3β enzyme ofapproximately 35 nM; and activity in Test (b) with an EC₅₀ versuscellular β-catenin accumulation in the nucleus of murine C3H/10T1/2cells of approximately 242 nM.

The pyrazine compound disclosed as Example 5 possesses activity in Test(a) with an IC₅₀ versus human GSK3β enzyme of approximately 21 nM; andactivity in Test (b) with an EC₅₀ versus cellular β-catenin accumulationin the nucleus of murine C3H/10T1/2 cells of approximately 902 nM.

The pyrazine compound disclosed as Example 6 possesses activity in Test(a) with an IC₅₀ versus human GSK3β enzyme of approximately 48 nM; andactivity in Test (b) with an EC₅₀ versus cellular β-catenin accumulationin the nucleus of murine C3H/10T1/2 cells of approximately 902 nM.

The pyrazine compound disclosed as Example 7 possesses activity in Test(a) with an IC₅₀ versus human GSK3β enzyme of approximately 80 nM; andactivity in Test (b) with an EC₅₀ versus cellular β-catenin accumulationin the nucleus of murine C3H/10T1/2 cells of approximately 3890 nM.

Conveniently, particular compounds of the invention possess activity atthe following concentrations or doses in the above tests (a) and (b):—

-   -   Test (a):—mean IC₅₀ versus human GSK3β enzyme in the range, for        example, 5 nM-40 nM    -   Test (b):—mean EC₅₀ versus cellular β-catenin accumulation in        murine C3H/10T1/2 cells, in the range, for example, 50 nM-300        nM.

For example, the pyrazine compound disclosed as Example 1 possessesactivity in Test (a) with an IC₅₀ versus human GSK3β enzyme ofapproximately 35 nM; and activity in Test (b) with an EC₅₀ versuscellular β-catenin accumulation in the nucleus of murine C3H/10T1/2cells of approximately 242 nM.

C) Osteogenic Differentiation and Assessment of Mineralisation In VitroMaterials and Methods

For in vitro osteogenesis, human adipose-derived stem cells (hADSC)(Invitrogen, UK; catalogue number R7788-115) were seeded into 12-wellplates at a density of 5000 cells/cm² and were cultured in a basal mediaof phenol red-free Dulbecco's Modified Eagles Medium (DMEM;Sigma-Aldrich, UK; catalogue number D1145) supplemented with 5% fetalbovine serum (FBS, Gibco, UK; catalogue number 10270) and 2 mM GlutaMax®(Gibco; catalogue number 35050). After overnight incubation, basal mediawas replaced and3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidetri-hydrochloride (solubilised in 100% DMSO and diluted in cell culturemedia to a final concentration of 0.2% DMSO) was added. In osteogenicpositive control wells, basal media was replaced with a differentiationmedia; phenol red-free DMEM supplemented with 5% FBS, 2 mM GlutaMax®, 50μg/ml L-ascorbic acid (Sigma-Aldrich; Catalogue no. A8960), 5 mMP3-glycerophosphate (Calbiochem, UK; catalogue number 35675), and 10 nMdexamethasone (Sigma-Aldrich; catalogue number D4902). The cultures wereincubated at 37° C. in 5% CO₂ with media and compound was replaced every3-4 days over a time-course of 24 days.

The formation of mineralized bone nodules was assessed during thetime-course using Alizarin Red S staining. At numerous times throughoutthe time-course, cells were fixed with 4% paraformaldehyde(Sigma-Aldrich; catalogue number P6148) for 20 min, followed by threewashes with phosphate buffered saline (PBS), without calcium chlorideand magnesium chloride, (Sigma-Aldrich; catalogue number D8537), beforebeing stained with 40 mM Alizarin Red S (Sigma-Aldrich; catalogue numberA5533) (pH4.2) for 20 min at room temperature. Plates were placed on arocking platform (50 rpm) during staining. After staining, cells werewashed three times with PBS, and three times with water. Cells wereimaged using a Leica M165FC stereomicroscope and associated LeicaApplication Suite imaging software (Leica Microsystems, UK).

The amount of mineralisation was quantified through the extraction ofthe Alizarin Red S stain from bone nodules using a cetylpyridiniumchloride extraction method, previously described (Gregory et al.,Analytical Biochemistry, 2004, 329: 77-84; Hwang et al., Stem Cells andDevelopment 17: 963-970, 2008). Briefly, Alizarin Red S stain wasextracted from bone nodules through the addition of 1 ml, to each well,of a cetylpyridium chloride buffer consisting of 10% cetylpyridiumchloride (Sigma-Aldrich; catalogue number C0732) in 10 mM Na₃PO₄(Sigma-Aldrich; catalogue number 342483) (pH7.0) and incubation for 1 hat 37° C. Following incubation, 200 μl aliquots were transferred to96-well format and the absorbance at 550 nm was determined using amicroplate spectrophotometer (SpectraMax Plus, Molecular Devices, USA).Data were expressed as mean absorbance at 550 nm±Standard error of mean(SEM). Differences between groups were examined for statisticalsignificance using one-way analysis of variance (ANOVA) followed byTukey's multiple comparison post hoc test. P<0.05 was consideredsignificant.

FIG. 1A: Images showing Alizarin red S staining of mineralised bonenodules in response to 300 nM3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidetri-hydrochloride compared with vehicle after 24 days of cell culture.Experiments were performed in triplicate.

FIG. 1B: Effect of 300 nM3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidetri-hydrochloride on bone mineralization, Alizarin red S stainingquantified using cetylpyridinium chloride extraction. Data are shown asmean absorbance at 550 nm and analysed using one-way ANOVA followed byTukey's multiple comparison post hoc test: n=9;***, P<0.001.

FIGS. 1A and 1B show that after 24 days of cell culture,3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidetri-hydrochloride significantly induced progenitor cell commitment andosteoblast differentiation as indicated through increased Alizarin red Sstaining relative to vehicle control.

d) Increased Bone Formation in Rats Compound Dosing, Blood Sampling andNecropsy

3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidetri-hydrochloride was formulated in water containing 0.5% HPMC((Hydroxypropyl)methyl cellulose; Catalogue number H7509, Sigma, Poole,UK) and 0.1% Tween 80 (v/v) (Catalogue number P8074, Sigma, Poole UK).Female Sprague Dawley rats (Charles River CRL:CD rats, Deal, Margate,UK) aged between 12 and 16 weeks (230-340 g) were dosed with eithercompound or vehicle with a 5 mL/kg volume administered by oral gavageusing straight steel (75 mm, 16 gauge) oral gavage feeding needles(Vet-Tech Solutions Ltd, Congleton, UK) between 7 and 10 am each day.Compound or vehicle was dosed at various doses (including 2.5, 5, 15 or50 mg/kg/day freebase doses equivalent to 3, 6, 18 and 59 mg/kg oftri-hydrochloride salt) once a day for seven to fourteen days. In somestudies a serum sample was taken prior to treatment to measure baselineserum bone biomarkers following overnight fasting. Blood was sampledfrom the tail vein and serum prepared by adding the blood to serum gelblood collection tubes (Microvette 500 Z-gel, catalogue number 20.1344,Sarstedt, Leicester, UK) and centrifuging the tubes for at 13000 rpm for5 min at 4° C. The serum samples were stored at −80° C. before biomarkeranalysis. Twenty fours hours after the last treatment dose, necropsieswere performed following termination by intraperitoneal injectionoverdose of pentobarbitone anesthetic (3 mL/kg of 200 mg/mL Euthatal,Merial Animal Health Ltd, Harlow, UK) in animals which had been fastedovernight. Following compound or vehicle treatment blood was taken fromthe descending aorta during necropsy in order to obtain terminal serumsamples for serum bone biomarker analysis. Blood was drawn into asterile 2.5 mL syringe through a 25 gauge×5/8 TW needle and decantedinto 2×0.5 mL serum gel blood collection tubes (Monovette 500 Z-gel,catalogue number 20.1344, Sarstedt, Leicester, UK), centrifuged andstored at −80° C. prior to analysis. The femurs and lumbar vertebraewere removed for histopathological processing and bone formationassessments. The left femur and the 4^(th) or 5^(th) lumbar vertebraewere processed for histopathology. For rapid decalcification of bones(for preparing haematoxylin and eosin stained sections), femurs werefixed in 10% neutral buffered formalin (Catalogue number PRC/R/4,Pioneer Chemicals, Colchester, UK) for 10 days followed by a 5 daydecalcification in 10% formic acid in water (Catalogue numberF/1850/PB17, Fisher Scientific, Loughborough, UK), changing thedecalcifying solution daily. Where immunohistochemistry was required,bones were fixed in 10% neutral buffered formalin for 10 days followedby decalcification with 12% EDTA (Diaminoethylene Tetra—Acetic Acid)plus 4% neutral buffered formalin (Catalogue number PRC/R/132, PioneerChemicals, Colchester, UK) at 50° C. for 7 weeks with the fluid changedevery week. Following decalcification, the femurs were washed for 2hours in running tap water and were processed by a standard overnightautomatic processing schedule using a series of alcohol, ethyl alcoholand xylene solutions before final wax embedding. Four μm thick sectionswere cut with a microtome, stained with haematoxylin and eosin andhistopathology assessment performed.

Evaluation of Bone Formation

The following techniques can be used to evaluate the effects ofcompounds on GSK3 inhibition and bone anabolism.

a) Histopathology

Histopathology changes in the rat femur sections were evaluated by lightmicroscopy and bone formation was assessed by changes in trabeculae andcortical bone.

After 7 or 14 days, oral administration of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidetri-hydrochloride, when dosed at 5 mg/kg, showed no differences inmorphology in the femur when compared to the relevant vehicle controlgroups.

After 7 days of oral administration of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidetri-hydrochloride, oral doses of 15 and 50 mg/kg were associated withdose related changes of increased osteoid and bone in both thesub-chondral region and in the cortical bone in the shaft. In addition,both doses were associated with evidence of osteoblast hypertrophy(suggestive of osteoblast activation). Additionally, at the 50 mg/kgdose, there was evidence of increased bone within the cancellous bonecompartment. This dose was also associated with histological evidence ofbone marrow cell mobilization and differentiation, notably by increasednumbers of immature (blast) osteoblasts within the cancellous bonecompartment and an increase in marrow cellularity.

After 14 days of oral administration of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidetri-hydrochloride, the histological effects of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidetri-hydrochloride were qualitatively similar to those seen following 7days of dosing but generally more pronounced and with evidence ofgreater bone marrow response to compound. Specifically, the 15 mg/kgdose of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidetri-hydrochloride showed evidence of bone formation in both thesub-chondral region and cortical region. Both findings were associatedwith osteoblast hypertrophy. The determination of bone formation wasfurther strengthened by the observation of focal bone masses in thesub-chondral region in 2 out of 10 animals. In the 50 mg/kg group, therewas a broader range of bone changes suggestive of bone formation inaddition to those noted above: increased osteoid and tide mark zonationin the sub-chondral region; increased osteoid in the cancellous bonecompartment and marked changes in bone marrow cellularity, specifically,expansion of a blast population which was morphologically consistentwith osteoblast expansion.

Taken together, these data provide morphological support to the conceptthat3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidetri-hydrochloride elicits bone formation in vivo.

b) Bone Densitometry

Bone mineral density was measured using peripheral quantitative computedtomography, pQCT (Jämsä et al. Bone, 1998, 23, 155-61, Tuukkanen et al.J Bone Mineral. Res. 2000, 15, 1905-11) using Norland Stratec XCTResearch SA+equipment with a Stratec software version 6.0 (NorlandStratec Medizintechnik, Birkenfeld, Germany). The pQCT measurements wereperformed ex vivo on each right femur with a voxel size of 0.07×0.07×0.5mm³. For the metaphyseal measurement, the site of CT scan was at thedistal end of femur, at 25% of total bone length from the distalarticular surface. The volumetric mineral density of the total bone(metaphysis total bone mineral density-BMD) was determined as mg/cm³.

Oral administration of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidetri-hydrochloride, dosed orally once a day for 14 consecutive days at 15mg/kg in female CRL:CD rats, resulted in an 6.6% increase in right femurmetaphysis total BMD compared with vehicle treated rats. Total BMD infemur from rats treated with3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidehydrochloride was 814.41+15 mg/cm³ or vehicle was 764.23±16 mg/cm³(mean±SEM). These results indicate that oral administration of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidetri-hydrochloride stimulates bone formation.

Bone Biomarker Evaluation

Bone biomarkers were determined by enzyme immunoassays (EIA) assay kitsfor N-terminal propeptide of type I procollagen (P1NP) and immunofixedenzyme activity assay kits for osteoclast-derived tartrate-resistantacid phosphatase form 5b (TRAcP-5b) specific for rat serum biomarkerquantification. Assay kits were purchased from Immunodiagnostic SystemsLtd (IDS Ltd, Boldon, UK) and used as per the manufacturer'sinstructions, to determine N-terminal propeptide of type I procollagen(P1NP-product code AC-33F1) and osteoclast-derived tartrate-resistantacid phosphatase form 5b (TRAcP-5b—product code SB-TR102) levels inserum.

PINP Assay

The P1NP assay used was a competitive immunoassay where P1NP in ratserum competes with biotin labeled PINP protein for plate immobilizedantibody binding. Ninety six well assay plates supplied pre-coated withpolyclonal rabbit anti-P1NP were incubated shaking at 700 rpm on a plateshaker for 1 hour at room temperature, with either 50 μL manufacturerssupplied standards, or 5 μL serum plus 45 μL supplied assay samplediluent, plus 50 μL solution P1NP biotin solution supplied whichcontained biotin labeled P1NP protein. Each well of the assay plate waswashed manually with 300 μL of supplied wash buffer three times, thewash buffer fully removed, and 150 μL of supplied enzyme conjugate(containing avidin linked to horseradish peroxidase) incubated for 30min at room temperature. The assay plates were washed 3 times with washbuffer and 150 μL supplied TMB solution containing an aqueousformulation of tetramethylbenzidine and hydrogen peroxide incubated for30 min at room temperature before the addition of 50 μL supplied 0.5MHCl to stop the reaction. The absorbance of all wells was determined at450 nm with a reference absorbance of 650 nm using a Molecular DevicesSpectromax-Plus spectrophotometer. The concentration of P1NP in serumsamples was determined in ng/mL by using standard regression analysisagainst the standard curve.

TRAcP-5b Assay

The TRAcP-5b assay was carried out in 96 well assay plates suppliedpre-coated with polyclonal anti-mouse IgG. Each well was incubated with100 μL anti Rat TRAcP-5b antibody for 60 min, shaking at 950 rpm on aplate shaker at room temperature. Each well of the assay plate waswashed manually with 300 μL of supplied wash buffer four times with anautomatic 1200 μL multichannel pipette. The wash buffer was tapped outand either 100 μL of supplied standards or 25 μL serum samples with 75μL 0.9% NaCl solution (Fresenius Kabi Ltd, Runcorn, UK, productcode—2295121E) were incubated with 50 μL supplied releasing reagent for1 hour shaking at room temperature. Each well of the assay plate waswashed manually with 300 μL of supplied wash buffer five times. The washbuffer was fully removed and 100 μL of supplied and freshly preparedpNPP (p-nitrophenyl phosphate) substrate solution was incubated for 1hour at 37° C. before the addition of 25 μL 0.32M NaOH was added to stopthe reaction. The absorbance of all wells was determined at 405 nm usinga Molecular Devices Spectromax-Plus spectrophotometer. The concentrationof TRAcP-5b in serum samples was determined in U/L by using standardregression analysis against the standard curve.

FIG. 2. shows serum P1NP levels following 7 days of oral dosing with3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidetri-hydrochloride in rats

FIG. 3. shows serum TRAcP-5b levels following 7 days of oral dosing with3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidetri-hydrochloride in rats

The data shown in FIG. 2 and FIG. 3 demonstrate that3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidetri-hydrochloride, dosed orally once a day for 7 consecutive days infemale CRL:CD rats, produced an increase in serum P1NP levels (marker ofbone formation) and a reduction in serum TRAcP-5b levels (marker of boneresorption). These results indicate that oral administration of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidetri-hydrochloride stimulates bone formation.

The following assay was used to measure the ability of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidetri-hydrochloride to inhibit CYP3A4 in recombinant human enzymes.

Assay

The ability of certain compounds to inhibit CYP3A4 in membranes wasdetermined from the inhibition of midazolam oxidation to 1′-hydroxymidazolam. Using midazolam, CYP3A4 catalyses the formation of1′-hydroxymidazolam, which was measured by LC MS/MS. Inhibition ofCYP3A4 resulted in a decrease in the rate of 1′-hydroxymidazolamproduction relative to control. Employing an automated assay (full IC₅₀)using the Automation Platform, up to thirteen compounds were run perplate as well as ketoconazole as a standard inhibitor of CYP3A4.

Incubation

The incubations were carried out in 0.1 M phosphate buffer (pH 7.4 at37° C.) containing DMSO (1%), midazolam (2.5 μM), phenacetin (25.7 μM),diclofenac (1.8 μM), S-mephenyloin (30.5 μM), bufuralol (8.8 μM), NADPH(1 mM), E. coli expressed 3A4 membranes (5 pmol/ml), 1A2 membranes (25pmol/ml), 2C9 membranes (5 pmol/ml), 2C19 membranes (2.5 pmol/ml), 2D6membranes (5 pmol/ml), and the test inhibitors ketoconazole,α-napthoflavone, sulphaphenazole, tranylcypromine and quinidine.Phenacetin and diclofenac were purchased from Sigma-Aldrich (Poole,dorset, UK). S-mephenyloin, bufuralol (hydrochloride salt) and midazolam(hydrochloride salt) were purchased from Ultrafine Chemicals(Manchester, UK). Recombinant expressed CYP enzymes were purchased fromCypex (Dundee, UK).

The assay was run on a robotic sample processor. The incubations werecarried out in micro titre plates. The assay is started by the additionof NADPH (reduced Nicotinamide Adenine Dinucleotide Phosphate), thereagents mixed and the plate pre-incubated. The plate was then incubatedfor 10 min in the hotel incubator. The reaction was terminated with theaddition of MeOH (1:1 vol/vol). The samples were centrifuged,transferred to a clean plate and analysed by LC MS/MS, which wasconducted using an Applied Biosystems—Sciex API4000 mass spectrometer,with Agilent HP1100 binary solvent delivery pumps, and CTC HTS-PALautosampler. Mobile phase consisted of Methanol and water with formicacid (0.1%) as modifier. The column used was a Phenomenex Max RP 50×2 mm(4 μm particle size). A gradient system was used, starting at 3% organicmobile phase for 0.25 min, raising to 100% organic at 1.75 min. Totalrun time was 2.5 min, with a flow rate of 0.6 ml/min. 10 μL was injectedonto column). The formation of product (1′-hydroxymidazolam) wasmonitored.

Inhibitor Concentrations

The recommended concentrations for the test compounds were 50, 15, 5,1.5, 0.5, 0.15 μM. A 5 mM stock of test inhibitor in DMSO was necessaryto achieve these concentrations. Ketoconazole was used as a standardinhibitor and was incubated at 0.09-0.0003 μM.

Data Analysis

Rates of reaction were calculated for each reaction by measuring MS/MSarea units. Data analysis was performed by linearising the data using apseudo Hill plot and utilizing an automated spreadsheet.

The degree of inhibition of CYP3A4 was calculated and expressed as anIC₅₀ value. The IC₅₀ value is the concentration of test compound thatinhibited 50% of CYP 3A4 activity. The estimated IC₅₀ value in the aboveassay for3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidetri-hydrochloride was >50 μM, indicating that the compound has neglibleactivity against CYP3A4.

The following assay was used to measure the activity of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidetri-hydrochloride against the cardiac Na⁺ channel, hNav1.5.

Cell Culture and Preparation

The hNav1.5-expressing Chinese hamster ovary K1 (CHO) cells used hasbeen described previously (Persson, F. et al, (2005), Journal ofCardiovascular Electrophysiology, 16, 329-341). These cells were grownto semi-confluence at 37° C. in a humidified environment (5% CO2) inF-12 Ham medium containing Glutamax™, 10% foetal calf serum and 1 mg/mlG418 (all Invitrogen). Prior to use, the monolayer was washed using apre-warmed (37° C.) 3 ml aliquot of Versene 1:5000 (Invitrogen). Afteraspiration of this solution the flask was incubated at 37° C. with afurther 3 ml of Versene 1:5000 for a period of 6 min. Cells were thendetached from the bottom of the flask by gentle tapping and 10 ml ofDulbecco's Phosphate-Buffered Saline containing calcium (0.9 mM) andmagnesium (0.5 mM) (PBS; Invitrogen) was then added to the flask andaspirated into a 15 ml centrifuge tube prior to centrifugation (50 g,for 4 min). The resulting supernatant was discarded and the pelletgently re-suspended in 3 ml of PBS. A 0.5 ml aliquot of cell suspensionwas removed and the number of viable cells (based on trypan blueexclusion) was determined in an automated cell counter (Cedex;Innovatis) so that the cell re-suspension volume could be adjusted withPBS to give the desired final cell concentration. For IonWorks Quattroexperiments, the final cell concentration used was 1,000,000 cells/ml.

Electrophysiology for IonWorks

The principles and operation of the IonWorks HT device have beendescribed by Schroeder et al. (Schroeder, et al (2003), Ionworks HT: anew high-throughput electrophysiology measurement platform. Journal ofBiomolecular Screening, 8, 50-64.2003). Briefly, the technology is basedon a 384-well plate (PatchPlate) in which a recording is attempted ineach well by using suction to position and hold a cell on a small holeseparating two isolated fluid chambers. Once sealing has taken place,the solution on the underside of the PatchPlate is changed to onecontaining amphotericin B. This permeabilises the patch of cell membranecovering the hole in each well and, in effect, allows a perforated,whole-cell patch clamp recording to be made from a single cell. TheIonWorks Quattro device uses the same technology except that recordingare made from up to 64 cells per well. PBS (Invitrogen) was used as theextracellular solution with the following composition (in mM): 138 NaCl,2.7 KCl, 0.9 CaCl2, 0.5 MgCl2, 1.5 KH2PO4, and 8.1 Na2HPO4. The“internal” solution (used during the seal formation process) compositionwas (in mM): Kgluconate 100, KCl 40, MgCl2 3.2, EGTA 3 and HEPES 5 (allSigma-Aldrich; pH 7.25-7.30 using 10 M KOH). The antibiotic containingsolution composition was (in mM): KCl 140, EGTA 1, MgCl2 1 and HEPES 20(pH 7.25-7.30 using 10 M KOH) plus 100 μg/ml of amphotericin B(Sigma-Aldrich). Each compound plate was laid-out in 12 columns toenable ten, 8-point concentration-effect curves to be constructed; theremaining two columns on the plate were taken up with vehicle (finalconcentration 0.33% DMSO), to define the assay baseline, and asupra-maximal blocking concentration of flecamide (final concentration316 μM) to define the 100% inhibition level. hNav1.5 currents weremeasured before and after approximately 3.5 min incubation with eachtest compound. In this way, non-cumulative concentration-effect curvescould be produced where, providing the acceptance criteria were achievedin a sufficient percentage of wells (see below). All recordings weremade at room temperature.

The pre- and post-compound hNav1.5 currents were evoked by a singlevoltage train consisting of a 15 s period holding at −90 mV, a 160 msstep to −100 mV (to obtain an estimate of leak current), a 100 ms stepback to −90 mV, followed by a series of 8 pulses to 0 mV for 50 ms froma holding potential of −90 mV applied at 3 Hz. (A stimulation frequencyof 3 Hz was chosen as it simulates the normal upper range of heart rateseen in humans—i.e. 180 beats per minute). In between the pre- andpost-compound voltage pulses there was no clamping of the membranepotential. Currents were leak-subtracted based on the estimate ofcurrent evoked during the step to −100 mV at the start of the voltagepulse protocol. The current signal was sampled at 2.5 kHz.

Pre- and post-scan hNav1.5 current amplitude was measured from theleak-subtracted traces by the IonWorks™ software by taking the averagecurrent during the end of the depolarising pulse to 0 mV (baselinecurrent) and subtracting this from the peak inward current. Theacceptance criteria for the currents evoked in each well were: pre-scanseal resistance >30 MΩ (Quattro), pre-scan Na+ current amplitude morenegative than −150 pA; post-scan seal resistance >30 MΩ (Quattro). Thedegree of inhibition of the hNav1.5 current was assessed by dividing thepost-scan hNav1.5 current by the respective pre-scan hNav1.5 current foreach well and for each of the eight test pulses.

The IC₅₀ value is the concentration of test compound that inhibited 50%of hNav1.5 activity. The IC₅₀ for3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidetri-hydrochloride was 21.2 μM, indicating that it does not displaypotent inhibitory activity against the cardiac Na⁺ channel, hNav1.5.

3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate was also tested in an in vivo rat genotoxicitymicronucleus study. The objective of this study was to investigatewhether two oral doses of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate increased the frequency of micronucleated immatureerythrocytes (MIE) in rat bone marrow.

3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate was administered to groups of 7 male Wistar Han rats,approximately 10 weeks old at dosing. Groups were given two doses of 0,25, 125 and 250 mg/kg free-base equivalent (0, 59.5, 297 and 595umol/kg) 24 hours apart and sampled for bone marrow analysis 24 hoursafter the second dose. Control rats were dosed with vehicle, 0.5% w/vhydroxypropyl methylcellulose and 0.1% w/v polysorbate 80 in phosphatebuffered saline. The highest dose was the maximum tolerated.

Animals were sacrificed 24 hours after the second dose and bone marrowsmears prepared, fixed and stained with acridine orange. For eachanimal, 2000 immature erythrocytes (1E) were scored for the incidence ofmicronucleated cells (MIE) and the ratio of IE to mature erythrocytes(E) was determined in 1000 cells. In order to act as a quality controlprocedure, 6 slides from the bone-marrow of rats treated withcyclophosphamide at mg/kg (72 umol/kg) in a previous study were includedwith the slides from the treated animals prior to staining and coding.

There were no biologically or statistically significant increases in theincidence of MIE above the vehicle control after two oral doses of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate. The IE:E ratio was similar in the treated and vehiclecontrol group. As expected, the incidence of MIE in the slides from ratspreviously given cyclophosphamide was significantly increased comparedwith the vehicle control values.

In conclusion,3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate did not increase the incidence of micronucleated immatureerythrocytes in the bone marrow of male Wistar Han rats whenadministered as two oral doses up to 250 mg/kg free-base equivalent (595umol/kg).

3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidetri-hydrochloride was also found to exhibit other desirable properties,such as, for example, pharmaco-kinetic properties and a high aqueoussolubility (mean measured solubility of approximately 1128 mg/ml insodium phosphate buffer at pH 7.4, n=4).

According to a further aspect of the invention there is provided apharmaceutical composition, which comprises a pyrazine derivative of theFormula I, or a pharmaceutically-acceptable salt thereof, as definedhereinbefore in association with a pharmaceutically-acceptable diluentor carrier.

The compositions of the invention may be in a form suitable for oral use(for example as tablets, lozenges, hard or soft capsules, aqueous oroily suspensions, emulsions, dispersible powders or granules, syrups orelixirs), for topical use (for example as creams, ointments, gels, oraqueous or oily solutions or suspensions), for administration byinhalation (for example as a finely divided powder or a liquid aerosol),for administration by insufflation (for example as a finely dividedpowder) or for parenteral administration (for example as a sterileaqueous or oily solution for intravenous, subcutaneous, intraperitonealor intramuscular dosing) or as a suppository for rectal dosing.

The compositions of the invention may also be used in conjunction with asuitable device or implant for treating bone tissue.

The compositions of the invention may be obtained by conventionalprocedures using conventional pharmaceutical excipients, well known inthe art. Thus, compositions intended for oral use may contain, forexample, one or more colouring, sweetening, flavouring and/orpreservative agents.

The amount of active ingredient that is combined with one or moreexcipients to produce a single dosage form will necessarily varydepending upon the host treated and the particular route ofadministration. For example, a formulation intended for oraladministration to humans will generally contain, for example, from 1 mgto 1 g of active agent (more suitably from 1 to 250 mg, for example from1 to 100 mg) compounded with an appropriate and convenient amount ofexcipients which may vary from about 5 to about 98 percent by weight ofthe total composition.

The size of the dose for therapeutic or prophylactic purposes of acompound of the Formula I will naturally vary according to the natureand severity of the disease state, the age and sex of the animal orpatient and the route of administration, according to well knownprinciples of medicine.

In using a compound of the Formula I for therapeutic or prophylacticpurposes it will generally be administered so that a daily dose in therange, for example, 1 mg/kg to 100 mg/kg body weight is received, givenif required in divided doses. In general, lower doses will beadministered when a parenteral route is employed. Thus, for example, forintravenous administration, a dose in the range, for example, 1 mg/kg to25 mg/kg body weight will generally be used. Similarly, foradministration by inhalation, a dose in the range, for example, 1 mg/kgto 25 mg/kg body weight will be used. Oral administration is howeverpreferred, particularly in tablet form. Typically, unit dosage formswill contain about 10 mg to 0.5 g of a compound of this invention.

As stated above, it has been found that compounds of the Formula Idefined in the present invention, are well suited for inhibitingglycogen synthase kinase-3 (GSK3). Accordingly, said compounds of thepresent invention are expected to be useful in the prevention and/ortreatment of conditions associated with glycogen synthase kinase-3activity, i.e. the compounds may be used to produce an inhibitory effectof GSK3 in mammals, including human, in need of such prevention and/ortreatment.

As stated hereinbefore, inhibitors of GSK3 enzymes should be oftherapeutic value for prevention and/or treatment of various forms ofbone-related disorders or conditions, such as, for example osteoporosis(genetic, iatrogenic or generated through aging/hormone imbalance),fracture repair as a result of injury or surgery, chronic-inflammatorydiseases that result in bone loss such as for example rheumatoidarthritis, cancers that lead to bone lesions, such as for examplecancers of the breast, prostate and lung, multiple myeloma,osteosarcoma, Ewing's sarcoma, chondrosarcoma, chordoma, malignantfibrous histiocytoma of the bone, fibrosarcoma of the bone, cancerinduced bone disease, iatrogenic bone disease, benign bone disease andPaget's disease.

According to an aspect of the invention there is provided a pyrazinederivative of the Formula I, or a pharmaceutically acceptable saltthereof, as defined hereinbefore for use as a medicament in awarm-blooded animal such as man.

According to a further aspect of the invention, there is provided apyrazine derivative of the Formula I, or a pharmaceutically acceptablesalt thereof, as defined hereinbefore for use in prevention and/ortreatment of conditions associated with GSK3 in a warm-blooded animalsuch as man.

According to a further aspect of the invention, there is provided theuse of a pyrazine derivative of the Formula I, or a pharmaceuticallyacceptable salt thereof, as defined hereinbefore for prevention and/ortreatment of conditions associated with GSK3 in a warm-blooded animalsuch as man.

According to a further feature of this aspect of the invention there isprovided the use of a pyrazine derivative of the Formula I, or apharmaceutically acceptable salt thereof, as defined hereinbefore in themanufacture of a medicament for use in the prevention and/or treatmentof conditions associated with GSK3 in a warm-blooded animal such as man.

According to a further feature of this aspect of the invention there isprovided a method for prevention and/or treatment of conditionsassociated with GSK3 in a warm blooded animal, such as man, in need ofsuch treatment which comprises administering to said animal an effectiveamount of a pyrazine derivative of the Formula I, or a pharmaceuticallyacceptable salt thereof, as defined hereinbefore.

According to a further aspect of the invention, there is provided apyrazine derivative of the Formula I, or a pharmaceutically acceptablesalt thereof, as defined hereinbefore for use in the prevention of boneloss in a warm-blooded animal such as man.

According to a further aspect of the invention, there is provided theuse of a pyrazine derivative of the Formula I, or a pharmaceuticallyacceptable salt thereof, as defined hereinbefore in the prevention ofbone loss in a warm-blooded animal such as man.

According to a further feature of this aspect of the invention there isprovided the use of a pyrazine derivative of the Formula I, or apharmaceutically acceptable salt thereof, as defined hereinbefore in themanufacture of a medicament for use in the in the prevention of boneloss effect in a warm-blooded animal such as man.

According to a further feature of this aspect of the invention there isprovided a method for preventing bone loss in a warm blooded animal,such as man, in need of such treatment which comprises administering tosaid animal an effective amount of a pyrazine derivative of the FormulaI, or a pharmaceutically acceptable salt thereof, as definedhereinbefore.

According to a further aspect of the invention there is provided apyrazine derivative of the Formula I, or a pharmaceutically acceptablesalt thereof, as defined hereinbefore for use in the prevention and/ortreatment of those bone-related disorders or conditions which aresensitive to inhibition of GSK-3 enzymes in a warm-blooded animal suchas man.

According to a further feature of this aspect of the invention there isprovided the use of a pyrazine derivative of the Formula I, or apharmaceutically acceptable salt thereof, as defined hereinbefore in themanufacture of a medicament for use in the prevention and/or treatmentof those bone-related disorders or conditions which are sensitive toinhibition of GSK-3 enzymes in a warm-blooded animal such as man.

According to a further feature of this aspect of the invention there isprovided a method for the prevention and/or treatment of thosebone-related disorders or conditions which are sensitive to inhibitionof GSK-3 enzymes, which comprises administering to a warm-blooded animalsuch as man.an effective amount of a pyrazine derivative of the FormulaI, or a pharmaceutically acceptable salt thereof, as definedhereinbefore.

According to a further feature of the invention there is provided apyrazine derivative of the Formula I, or a pharmaceutically acceptablesalt thereof, as defined hereinbefore for use in the prevention and/ortreatment of osteoporosis (genetic, iatrogenic or generated throughaging/hormone imbalance), fracture repair as a result of injury orsurgery, chronic-inflammatory diseases that result in bone loss such asfor example rheumatoid arthritis, bone lesions in cancers that lead tosuch bone lesions, such as for example cancers of the breast, prostateand lung, multiple myeloma, osteosarcoma, Ewing's sarcoma,chondrosarcoma, chordoma, malignant fibrous histiocytoma of the bone,fibrosarcoma of the bone, cancer induced bone disease, iatrogenic bonedisease, benign bone disease and Paget's disease in a warm-bloodedanimal such as man.

According to a further feature of this aspect of the invention there isprovided the use of a pyrazine derivative of the Formula I, or apharmaceutically acceptable salt thereof, as defined hereinbefore in themanufacture of a medicament for use in the prevention and/or treatmentof osteoporosis (genetic, iatrogenic or generated through aging/hormoneimbalance), fracture repair as a result of injury or surgery,chronic-inflammatory diseases that result in bone loss such as forexample rheumatoid arthritis, bone lesions in cancers that lead to suchbone lesions, such as for example cancers of the breast, prostate andlung, multiple myeloma, osteosarcoma, Ewing's sarcoma, chondrosarcoma,chordoma, malignant fibrous histiocytoma of the bone, fibrosarcoma ofthe bone, cancer induced bone disease, iatrogenic bone disease, benignbone disease and Paget's disease in a warm-blooded animal such as man.

According to a further feature of this aspect of the invention there isprovided the use of a pyrazine derivative of the Formula I, or apharmaceutically acceptable salt thereof, as defined hereinbefore in themanufacture of a medicament for use in the prevention and/or treatmentof osteoporosis (genetic, iatrogenic or generated through aging/hormoneimbalance), fracture repair as a result of injury or surgery,chronic-inflammatory diseases that result in bone loss such as forexample rheumatoid arthritis, bone lesions in cancers that lead to suchbone lesions, such as for example cancers of the breast, prostate andlung, multiple myeloma, osteosarcoma, Ewing's sarcoma, chondrosarcoma,chordoma, malignant fibrous histiocytoma of the bone, fibrosarcoma ofthe bone, cancer induced bone disease, iatrogenic bone disease, benignbone disease and Paget's disease in a warm-blooded animal such as man.

According to a further feature of this aspect of the invention there isprovided a method for preventing and/or treating osteoporosis (genetic,iatrogenic or generated through aging/hormone imbalance), fracturerepair as a result of injury or surgery, chronic-inflammatory diseasesthat result in bone loss such as for example rheumatoid arthritis, bonelesions in cancers that lead to such bone lesions, such as for examplecancers of the breast, prostate and lung, multiple myeloma,osteosarcoma, Ewing's sarcoma, chondrosarcoma, chordoma, malignantfibrous histiocytoma of the bone, fibrosarcoma of the bone, cancerinduced bone disease, iatrogenic bone disease, benign bone disease andPaget's disease in a warm blooded animal such as man that is in need ofsuch treatment which comprises administering an effective amount of apyrazine derivative of the Formula I, or a pharmaceutically acceptablesalt thereof, as defined hereinbefore.

A pyrazine compound of Formula I as hereinbefore defined could be usefulin primary and secondary ostopeorosis, where primary osteoporosisincludes postmenopausal osteoporosis and senile osteoporosis in both menand women, and secondary osteoporosis includes cortison inducedosteoporosis, as well as any other type of induced secondaryosteoporosis, are included in the term osteoporosis. A pyrazine compoundof Formula I may be administered locally or systemically, in differentformulation regimes, to treat these conditions.

Accordingly, in a further feature of the invention there is provided apyrazine derivative of the Formula I, or a pharmaceutically acceptablesalt thereof, as defined hereinbefore for use in the treatment ofosteoporosis (genetic, iatrogenic or generated through aging/hormoneimbalance) in a warm-blooded animal such as man.

According to a further feature of this aspect of the invention there isprovided the use of a pyrazine derivative of the Formula I, or apharmaceutically acceptable salt thereof, as defined hereinbefore in themanufacture of a medicament for use in the treatment of osteoporosis(genetic, iatrogenic or generated through aging/hormone imbalance) in awarm-blooded animal such as man.

According to a further feature of this aspect of the invention there isprovided the use of a pyrazine derivative of the Formula I, or apharmaceutically acceptable salt thereof, as defined hereinbefore in themanufacture of a medicament for use in the treatment of osteoporosis(genetic, iatrogenic or generated through aging/hormone imbalance) in awarm-blooded animal such as man.

According to a further feature of this aspect of the invention there isprovided a method for treating osteoporosis (genetic, iatrogenic orgenerated through aging/hormone imbalance) in a warm blooded animal suchas man that is in need of such treatment which comprises administeringan effective amount of a chromenone derivative of the Formula I, or apharmaceutically acceptable salt thereof, as defined hereinbefore.

The promotion and increasing of bone formation and/or bone mineraldensity makes a compound of the Formula I as hereinbefore defined,suitable for reducing the incidence of fracture, to reduce the rate offracture and/or increase the rate of fracture healing, to increasecancellous bone formation and/or new bone formation in mammals. The useto promote and increase new bone formation may be in connection withsurgery. This invention can be used during surgery, where the treatingsurgeon will place the invention locally in an appropriate formulation,near the deficient bone and/or in the body cavity. The bone may forinstance have been broken, and utilizing the invention as described andclaimed herein will then be placed in or near the fracture during openfracture repair. In some instances bone pieces may be missing (e.g.after tumour removal or severe casualties), and utilizing the inventionas described and claimed herein will then be placed near the site ofconstructive bone surgery.

Accordingly, in a further feature of the invention there is provided apyrazine derivative of the Formula I, or a pharmaceutically acceptablesalt thereof, as defined hereinbefore for use in promoting boneformation in a warm-blooded animal such as man.

According to a further feature of this aspect of the invention there isprovided the use of a pyrazine derivative of the Formula I, or apharmaceutically acceptable salt thereof, as defined hereinbefore in themanufacture of a medicament for use in promoting bone formation in awarm-blooded animal such as man.

According to a further feature of this aspect of the invention there isprovided the use of a pyrazine derivative of the Formula I, or apharmaceutically acceptable salt thereof, as defined hereinbefore in themanufacture of a medicament for use in promoting bone formation in awarm-blooded animal such as man.

According to a further feature of this aspect of the invention there isprovided a method for promoting bone formation in a warm blooded animalsuch as man that is in need of such treatment which comprisesadministering an effective amount of a pyrazine derivative of theFormula I, or a pharmaceutically acceptable salt thereof, as definedhereinbefore.

According to a further feature of the invention there is provided apyrazine derivative of the Formula I, or a pharmaceutically acceptablesalt thereof, as defined hereinbefore for use in increasing bone mineraldensity in a warm-blooded animal such as man.

According to a further feature of this aspect of the invention there isprovided the use of a pyrazine derivative of the Formula I, or apharmaceutically acceptable salt thereof, as defined hereinbefore in themanufacture of a medicament for use in increasing bone mineral densityin a warm-blooded animal such as man.

According to a further feature of this aspect of the invention there isprovided the use of a pyrazine derivative of the Formula I, or apharmaceutically acceptable salt thereof, as defined hereinbefore in themanufacture of a medicament for use in increasing bone mineral densityin a warm-blooded animal such as man.

According to a further feature of this aspect of the invention there isprovided a method for increasing bone mineral density in a warm bloodedanimal such as man that is in need of such treatment which comprisesadministering an effective amount of a pyrazine derivative of theFormula I, or a pharmaceutically acceptable salt thereof, as definedhereinbefore.

According to a further feature of the invention there is provided apyrazine derivative of the Formula I, or a pharmaceutically acceptablesalt thereof, as defined hereinbefore for use in reducing the incidenceof fracture and/or increasing the rate of fracture healing and/orincreasing the strength of a healed fracture in a warm-blooded animalsuch as man.

According to a further feature of this aspect of the invention there isprovided the use of a pyrazine derivative of the Formula I, or apharmaceutically acceptable salt thereof, as defined hereinbefore in themanufacture of a medicament for use in reducing the incidence offracture and/or increasing the rate of fracture healing and/orincreasing the strength of a healed fracture in a warm-blooded animalsuch as man.

According to a further feature of this aspect of the invention there isprovided the use of a pyrazine derivative of the Formula I, or apharmaceutically acceptable salt thereof, as defined hereinbefore in themanufacture of a medicament for use in reducing the incidence offracture and/or increasing the rate of fracture healing and/orincreasing the strength of a healed fracture in a warm-blooded animalsuch as man.

According to a further feature of this aspect of the invention there isprovided a method for reducing the incidence of fracture and/orincreasing the rate of fracture healing and/or increasing the strengthof a healed fracture in a warm blooded animal such as man that is inneed of such treatment which comprises administering an effective amountof a pyrazine derivative of the Formula I, or a pharmaceuticallyacceptable salt thereof, as defined hereinbefore.

According to a further feature of the invention there is provided apyrazine derivative of the Formula I, or a pharmaceutically acceptablesalt thereof, as defined hereinbefore for use in prevention and/ortreatment of bone lesions in cancers such as for example cancers of thebreast, prostate and lung, multiple myeloma, osteosarcoma, Ewing'ssarcoma, chondrosarcoma, chordoma, malignant fibrous histiocytoma of thebone, fibrosarcoma of the bone, cancer induced bone disease, iatrogenicbone disease, benign bone disease and Paget's disease in a warm-bloodedanimal such as man.

According to a further feature of this aspect of the invention there isprovided the use of a pyrazine derivative of the Formula I, or apharmaceutically acceptable salt thereof, as defined hereinbefore in themanufacture of a medicament for use in prevention and/or treatment ofbone lesions in cancers such as for example cancers of the breast,prostate and lung, multiple myeloma, osteosarcoma, Ewing's sarcoma,chondrosarcoma, chordoma, malignant fibrous histiocytoma of the bone,fibrosarcoma of the bone, cancer induced bone disease, iatrogenic bonedisease, benign bone disease and Paget's disease in a warm-bloodedanimal such as man.

According to a further feature of this aspect of the invention there isprovided the use of a pyrazine derivative of the Formula I, or apharmaceutically acceptable salt thereof, as defined hereinbefore in themanufacture of a medicament for use in prevention and/or treatment ofbone lesions in cancers such as for example cancers of the breast,prostate and lung, multiple myeloma, osteosarcoma, Ewing's sarcoma,chondrosarcoma, chordoma, malignant fibrous histiocytoma of the bone,fibrosarcoma of the bone, cancer induced bone disease, iatrogenic bonedisease, benign bone disease and Paget's disease in a warm-bloodedanimal such as man.

According to a further feature of this aspect of the invention there isprovided a method for prevention and/or treatment of bone lesions incancers such as for example cancers of the breast, prostate and lung,multiple myeloma, osteosarcoma, Ewing's sarcoma, chondrosarcoma,chordoma, malignant fibrous histiocytoma of the bone, fibrosarcoma ofthe bone, cancer induced bone disease, iatrogenic bone disease, benignbone disease and Paget's disease in a warm blooded animal such as manthat is in need of such treatment which comprises administering aneffective amount of a pyrazine derivative of the Formula I, or apharmaceutically acceptable salt thereof, as defined hereinbefore.

According to a further feature of the invention there is provided apyrazine derivative of the Formula I, or a pharmaceutically acceptablesalt thereof, as defined hereinbefore for use in prevention and/ortreatment of bone lesions in multiple myeloma in a warm-blooded animalsuch as man.

According to a further feature of this aspect of the invention there isprovided the use of a pyrazine derivative of the Formula I, or apharmaceutically acceptable salt thereof, as defined hereinbefore in themanufacture of a medicament for use in prevention and/or treatment ofbone lesions in bone lesions in multiple myeloma in a warm-bloodedanimal such as man.

According to a further feature of this aspect of the invention there isprovided a method for prevention and/or treatment of bone lesions inbone lesions in multiple myeloma in a warm blooded animal such as manthat is in need of such treatment which comprises administering aneffective amount of a pyrazine derivative of the Formula I, or apharmaceutically acceptable salt thereof, as defined hereinbefore.

According to a further feature of the invention there is provided apyrazine derivative of the Formula I, or a pharmaceutically acceptablesalt thereof, as defined hereinbefore for use in the prevention ofskeletal-related events in a warm-blooded animal such as man with bonelesions as a result of cancers such as for example cancers of thebreast, prostate and lung, multiple myeloma, osteosarcoma, Ewing'ssarcoma, chondrosarcoma, chordoma, malignant fibrous histiocytoma of thebone, fibrosarcoma of the bone, cancer induced bone disease, iatrogenicbone disease, benign bone disease and Paget's disease.

According to a further feature of this aspect of the invention there isprovided the use of a pyrazine derivative of the Formula I, or apharmaceutically acceptable salt thereof, as defined hereinbefore in themanufacture of a medicament for use in the prevention ofskeletal-related events in a warm-blooded animal such as man with bonelesions as a result of cancers such as for example cancers of thebreast, prostate and lung, multiple myeloma, osteosarcoma, Ewing'ssarcoma, chondrosarcoma, chordoma, malignant fibrous histiocytoma of thebone, fibrosarcoma of the bone, cancer induced bone disease, iatrogenicbone disease, benign bone disease and Paget's disease.

According to a further feature of this aspect of the invention there isprovided a method for preventing skeletal-related events in awarm-blooded animal such as man with bone lesions as a result of cancerssuch as for example cancers of the breast, prostate and lung, multiplemyeloma, osteosarcoma, Ewing's sarcoma, chondrosarcoma, chordoma,malignant fibrous histiocytoma of the bone, fibrosarcoma of the bone,cancer induced bone disease, iatrogenic bone disease, benign bonedisease and Paget's disease that is in need of such treatment whichcomprises administering an effective amount of a pyrazine derivative ofthe Formula I, or a pharmaceutically acceptable salt thereof, as definedhereinbefore.

In addition to bone-related disorders or conditions, there is evidencethat GSK3 enzymes play a role in other diseases. Thus inhibitors of GSK3enzymes, are expected to be of value in the prevention and treatment ofa wide variety of diseases in addition to bone-related disorders orconditions. For example, inhibitors of GSK3 enzymes, are expected to beof value in the treatment of cancer. Indeed a particular pyrazinederivative of the Formula I, namely3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidetri-hydrochloride, has also been found to exhibit inhibitory activityagainst other kinases such as PIM3, CHK2 (checkpoint kinase 2) andDYRK1a (Dual Specificity Tyrosine Phosphorylated and Regulated Kinase1a), which are also believed to play a role in cancer.

Accordingly, a pyrazine derivative of the Formula I, or apharmaceutically acceptable salt thereof, should be of therapeutic valuefor treatment of the various forms of the disease of cancer comprisingsolid tumours such as carcinomas and sarcomas and the leukaemias andlymphoid malignancies. In particular, a pyrazine derivative of theFormula I, or a pharmaceutically acceptable salt thereof, should be oftherapeutic value for treatment of, for example, cancer of the breast,colorectum, lung (including small cell lung cancer, non-small cell lungcancer and bronchioalveolar cancer) and prostate, and of cancer of thebile duct, bone, bladder, head and neck, kidney, liver, gastrointestinaltissue, oesophagus, ovary, pancreas, skin, testes, thyroid, uterus,cervix and vulva, and of leukaemias (including ALL and CML), multiplemyeloma and lymphomas.

According to a further aspect of the invention, there is provided apyrazine derivative of the Formula I, or a pharmaceutically acceptablesalt thereof, as defined hereinbefore for use in the treatment of cancerin a warm blooded animal such as man.

According to a further aspect of the invention there is provided the useof a pyrazine derivative of the Formula I, or a pharmaceuticallyacceptable salt thereof, as defined hereinbefore in the manufacture of amedicament for use in the treatment of cancer in a warm blooded animalsuch as man.

According to a further feature of this aspect of the invention there isprovided a method for the treatment of cancer in a warm blooded animal,such as man, in need of such treatment which comprises administering tosaid animal an effective amount of a pyrazine derivative of the FormulaI, or a pharmaceutically acceptable salt thereof, as definedhereinbefore.

According to a further aspect of the invention, there is provided apyrazine derivative of the Formula I, or a pharmaceutically acceptablesalt thereof, as defined hereinbefore for use in the treatment of cancerselected from cancer of the breast, colorectum, lung (including smallcell lung cancer, non-small cell lung cancer and bronchioalveolarcancer) and prostate.

According to a further aspect of the invention there is provided the useof a pyrazine derivative of the Formula I, or a pharmaceuticallyacceptable salt thereof, as defined hereinbefore in the manufacture of amedicament for use in treatment of cancer selected from cancer of thebreast, colorectum, lung (including small cell lung cancer, non-smallcell lung cancer and bronchioalveolar cancer) and prostate in a warmblooded animal such as man.

According to a further feature of this aspect of the invention there isprovided a method for the treatment of cancer selected from cancer ofthe breast, colorectum, lung (including small cell lung cancer,non-small cell lung cancer and bronchioalveolar cancer) and prostate ina warm blooded animal, such as man, in need of such treatment whichcomprises administering to said animal an effective amount of a pyrazinederivative of the Formula I, or a pharmaceutically acceptable saltthereof, as defined hereinbefore.

In the context of the present specification, the term “therapy” or“treatment” also includes “prevention” unless there are specificindications to the contrary. The terms “therapeutic” and“therapeutically” should be construed accordingly.

In the context of the present specification, the term “disorder” alsoincludes “condition” unless there are specific indications to thecontrary.

As stated hereinbefore, the in vivo effects of a compound of the FormulaI may be exerted in part by one or more metabolites that are formedwithin the human or animal body after administration of a compound ofthe Formula I.

The anti-bone loss treatment defined hereinbefore may be applied as asole therapy or may involve, in addition to a compound of the invention,conventional surgery or radiotherapy or chemotherapy. Such chemotherapymay include one or more of the following categories of anti-tumouragents:—

(i) other antiproliferative/antineoplastic drugs and combinationsthereof, as used in medical oncology, such as alkylating agents (forexample cis-platin, oxaliplatin, carboplatin, cyclophosphamide, nitrogenmustard, melphalan, chlorambucil, busulphan, temozolamide andnitrosoureas); antimetabolites (for example gemcitabine and antifolatessuch as fluoropyrimidines like 5-fluorouracil and tegafur, raltitrexed,methotrexate, cytosine arabinoside, and hydroxyurea); antitumourantibiotics (for example anthracyclines like adriamycin, bleomycin,doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C,dactinomycin and mithramycin); antimitotic agents (for example vincaalkaloids like vincristine, vinblastine, vindesine and vinorelbine andtaxoids like taxol and taxotere and polokinase inhibitors); andtopoisomerase inhibitors (for example epipodophyllotoxins like etoposideand teniposide, amsacrine, topotecan and camptothecin);(ii) cytostatic agents such as antioestrogens (for example tamoxifen,fulvestrant, toremifene, raloxifene, droloxifene and iodoxyfene),antiandrogens (for example bicalutamide, flutamide, nilutamide andcyproterone acetate), LHRH antagonists or LHRH agonists (for examplegoserelin, leuprorelin and buserelin), progestogens (for examplemegestrol acetate), aromatase inhibitors (for example as anastrozole,letrozole, vorazole and exemestane) and inhibitors of 5α-reductase suchas finasteride;(iii) anti-invasion agents [for example c-Src kinase family inhibitorslike4-(6-chloro-2,3-methylenedioxyanilino)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5-tetrahydropyran-4-yloxyquinazoline(AZD0530; International Patent Application WO 01/94341),N-(2-chloro-6-methylphenyl)-2-{6-[4-(2-hydroxyethyl)piperazin-1-yl]-2-methylpyrimidin-4-ylamino}thiazole-5-carboxamide(dasatinib, BMS-354825; J. Med. Chem., 2004, 47, 6658-6661) andbosutinib (SKI-606), and metalloproteinase inhibitors like marimastat,inhibitors of urokinase plasminogen activator receptor function orantibodies to Heparanase];(iv) inhibitors of growth factor function: for example such inhibitorsinclude growth factor antibodies and growth factor receptor antibodies(for example the anti-erbB2 antibody trastuzumab [Herceptin™], theanti-EGFR antibody panitumumab, the anti-erbB1 antibody cetuximab[Erbitux, C225] and any growth factor or growth factor receptorantibodies disclosed by Stern et al. Critical reviews inoncology/haematology, 2005, Vol. 54, pp11-29); such inhibitors alsoinclude tyrosine kinase inhibitors, for example inhibitors of theepidermal growth factor family (for example EGFR family tyrosine kinaseinhibitors such asN-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-amine(gefitinib, ZD1839),N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine(erlotinib, OSI-774) and6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)-quinazolin-4-amine(CI 1033), erbB2 tyrosine kinase inhibitors such as lapatinib);inhibitors of the hepatocyte growth factor family; inhibitors of theinsulin growth factor family; inhibitors of the platelet-derived growthfactor family such as imatinib and/or nilotinib (AMN107); inhibitors ofserine/threonine kinases (for example Ras/Raf signalling inhibitors suchas farnesyl transferase inhibitors, for example sorafenib (BAY 43-9006),tipifarnib (R115777) and lonafarnib (SCH66336)), inhibitors of cellsignalling through MEK and/or AKT kinases, c-kit inhibitors, abl kinaseinhibitors, PI3 kinase inhibitors, Plt3 kinase inhibitors, CSF-1R kinaseinhibitors, IGF receptor (insulin-like growth factor) kinase inhibitors;aurora kinase inhibitors (for example AZD1152, PH739358, VX-680,MLN8054, R763, MP235, MP529, VX-528 AND AX39459) and cyclin dependentkinase inhibitors such as CDK2 and/or CDK4 inhibitors;(v) antiangiogenic agents such as those which inhibit the effects ofvascular endothelial growth factor, [for example the anti-vascularendothelial cell growth factor antibody bevacizumab (Avastin™) and forexample, a VEGF receptor tyrosine kinase inhibitor such as vandetanib(ZD6474), vatalanib (PTK787), sunitinib (SU11248), axitinib (AG-013736),pazopanib (GW 786034) and4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline(AZD2171; Example 240 within WO 00/47212), compounds such as thosedisclosed in International Patent Applications WO97/22596, WO 97/30035,WO 97/32856 and WO 98/13354 and compounds that work by other mechanisms(for example linomide, inhibitors of integrin αvβ3 function andangiostatin)];(vi) vascular damaging agents such as Combretastatin A4 and compoundsdisclosed in International Patent Applications WO 99/02166, WO 00/40529,WO 00/41669, WO 01/92224, WO 02/04434 and WO 02/08213;(vii) an endothelin receptor antagonist, for example zibotentan (ZD4054)or atrasentan;(viii) antisense therapies, for example those which are directed to thetargets listed above, such as ISIS 2503, an anti-ras antisense;(ix) a proteosome inhibitor such as for example, Velcade (bortezomib)(x) gene therapy approaches, including for example approaches to replaceaberrant genes such as aberrant p53 or aberrant BRCA1 or BRCA2, GDEPT(gene-directed enzyme pro-drug therapy) approaches such as those usingcytosine deaminase, thymidine kinase or a bacterial nitroreductaseenzyme and approaches to increase patient tolerance to chemotherapy orradiotherapy such as multi-drug resistance gene therapy; and(xi) immunotherapy approaches, including for example ex-vivo and in-vivoapproaches to increase the immunogenicity of patient tumour cells, suchas transfection with cytokines such as interleukin 2, interleukin 4 orgranulocyte-macrophage colony stimulating factor, approaches to decreaseT-cell anergy, approaches using transfected immune cells such ascytokine-transfected dendritic cells, approaches usingcytokine-transfected tumour cell lines and approaches usinganti-idiotypic antibodies.

According to this aspect of the invention there is provided acombination suitable for use in the treatment of bone-related disordersor conditions comprising a compound of formula I as defined hereinbeforeor a pharmaceutically acceptable salt thereof and any one of the antitumour agents listed under (i)-(xi) above.

Therefore in a further aspect of the invention there is provided acompound of Formula I or a pharmaceutically acceptable salt thereof incombination with an anti-tumour agent selected from one listed under(i)-(xi) herein above.

Herein, where the term “combination” is used it is to be understood thatthis refers to simultaneous, separate or sequential administration. Inone aspect of the invention “combination” refers to simultaneousadministration. In another aspect of the invention “combination” refersto separate administration. In a further aspect of the invention“combination” refers to sequential administration. Where theadministration is sequential or separate, the delay in administering thesecond component should not be such as to lose the beneficial effect ofthe combination.

According to a further aspect of the invention there is provided apharmaceutical composition which comprises a compound of Formula I or apharmaceutically acceptable salt thereof in combination with ananti-tumour agent selected from one listed under (i)-(xi) herein above,in association with a pharmaceutically acceptable diluent or carrier.

According to a further aspect of the invention there is provided apharmaceutical composition which comprises a compound of Formula I or apharmaceutically acceptable salt thereof in combination with ananti-tumour agent selected from one listed under (i)-(xi) herein above,in association with a pharmaceutically acceptable diluent or carrier foruse in treating bone-related disorders or conditions.

According to another feature of the invention there is provided the useof a compound of the Formula I or a pharmaceutically acceptable saltthereof in combination with an anti-tumour agent selected from onelisted under (i)-(xi) herein above, in the manufacture of a medicamentfor use in bone-related disorders or conditions in a warm-bloodedanimal, such as man.

Therefore in an additional feature of the invention, there is provided amethod of treating bone-related disorders or conditions in awarm-blooded animal, such as man, in need of such treatment whichcomprises administering to said animal an effective amount of a compoundof Formula I or a pharmaceutically acceptable salt thereof incombination with an anti-tumour agent selected from one listed under(i)-(xi) herein above.

According to a further aspect of the present invention there is provideda kit comprising a compound of Formula I or a pharmaceuticallyacceptable salt thereof in combination with an anti-tumour agentselected from one listed under (i)-(xi) herein above.

According to a further aspect of the present invention there is provideda kit comprising:

a) a compound of Formula I or a pharmaceutically acceptable salt thereofin a first unit dosage form;b) an anti-tumour agent selected from one listed under (i)-(xi) hereinabove; in a second unit dosage form; andc) container means for containing said first and second dosage forms.

The anti-bone loss treatment defined hereinbefore may be applied as asole therapy or may involve, in addition to a compound of the invention,combination with other pharmacologically active agent(s), such as forexample bisphosphonates, estrogen, SERMS (selective estrogen receptormodulators), RANKL (receptor activator of nuclear factor kB ligand)antagonists, calcitonins and oestogenic promoting agents such asparathyroid hormone or recombinant or synthetic parathyroid hormone.Examples of bisphosphonates include alendronate, clodronate,ibandronate, pamidronate, etidronate, risedronate, tiludronate andzoledronic acid. Examples of SERMS include raloxifene anddihydroraloxifene. An example of a RANKL antagonist is denosumab.Calcitonins include human and salmon calcitonins.

According to this aspect of the invention there is provided acombination suitable for use in the treatment of bone-related disordersor conditions comprising a compound of formula I as defined hereinbeforeor a pharmaceutically acceptable salt thereof and a pharmacologicallyactive agent, selected from a bisphosphonate, estrogen, a SERM, a RANKLantagonist, calcitonin and an oestogenic promoting agent.

According to a further aspect of the invention there is provided apharmaceutical composition which comprises a compound of Formula I or apharmaceutically acceptable salt thereof in combination with apharmacologically active agent, selected from a bisphosphonate,estrogen, a SERM, a RANKL antagonist, calcitonin and an oestogenicpromoting agent, in association with a pharmaceutically acceptablediluent or carrier.

According to a further aspect of the invention there is provided apharmaceutical composition which comprises a compound of Formula I or apharmaceutically acceptable salt thereof in combination with apharmacologically active agent, selected from a bisphosphonate,estrogen, a SERM, a RANKL antagonist, calcitonin and an oestogenicpromoting agent, in association with a pharmaceutically acceptablediluent or carrier for use in treating bone-related disorders orconditions.

According to another feature of the invention there is provided the useof a compound of the Formula I or a pharmaceutically acceptable saltthereof in combination with a pharmacologically active agent, selectedfrom a bisphosphonate, estrogen, a SERM, a RANKL antagonist, calcitoninand an oestogenic promoting agent, in the treatment of bone-relateddisorders or conditions in a warm-blooded animal, such as man.

Therefore in an additional feature of the invention, there is provided amethod of treating bone-related disorders or conditions in awarm-blooded animal, such as man, in need of such treatment whichcomprises administering to said animal an effective amount of a compoundof Formula I or a pharmaceutically acceptable salt thereof incombination with a pharmacologically active agent, selected from abisphosphonate, estrogen, a SERM, a RANKL antagonist, calcitonin and anoestogenic promoting agent.

Although the compounds of the Formula I are primarily of value astherapeutic agents for use in warm-blooded animals (including man), theyare also useful whenever it is required to inhibit the effects of GSK-3.Thus, they are useful as pharmacological standards for use in thedevelopment of new biological tests and in the search for newpharmacological agents.

The invention will now be illustrated in the following Examples inwhich, generally:

(i) operations were carried out at ambient temperature, i.e. in therange 17 to 25° C. and under an atmosphere of an inert gas such asnitrogen unless otherwise stated;

(ii) evaporations were carried out by rotary evaporation and work-upprocedures were carried out after removal of residual solids byfiltration;

(iii) flash chromatography purifications were performed on an automatedcombiflash companion (TELEDYNE, ISCO, USA), using prepacked Agela normalphase Si60 silica cartridges obtained from Agela technologies;

(iv) preparative chromatography was performed on a SHIMADZU instrument(LC-8A) fitted with a ZMD or ZQ ESCi mass spectrometers and a Lunareverse-phase column (C-18, 5 microns silica, 50 mm diameter, 250 mmlength, flow rate of 80 ml/minute) using decreasingly polar mixtures ofwater (containing 0.1% TFA) and acetonitrile as eluent, or RPHPLC(Reversed Phase Preparative High Performance Liquid Chromatography)using Waters Symmetry C8, Xterra or Phenomenex Gemini columns usingacetonitrile and either aqueous ammonium acetate, ammonia, formic acidor trifluoroacetic acid as buffer where appropriate;

(v) yields, where present, are not necessarily the maximum attainable;

(vi) in general, the structures of end-products of the Formula I wereconfirmed by nuclear magnetic resonance (NMR) spectroscopy; NMR chemicalshift values were measured on the delta scale [proton magnetic resonancespectra were determined using a Varian or Bruker Avance 400 (400 MHz)instrument; measurements were taken at ambient temperature unlessotherwise specified; the following abbreviations have been used: s,singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dd, doubletof doublets; ddd, doublet of doublet of doublet; dt, doublet oftriplets; bs, broad signal;

(vii) in general, end-products of the Formula I were also characterisedby mass spectroscopy following liquid chromatography (LCMS); LCMS wascarried out using an SHIMADZU 20 AB fitted with SHIMADZU 2010EV massspectrometer with a Xtimate 3 um C-18 column (2.1×30 mm) or a Shim-packXR-ODS C-18 column (3.0×30 mm) at either: (a) a flow rate of 1.2 ml/min,using a solvent system of 10% A+90% B to 80% A+20% B over 3 minutes,where A=0.01875% TFA in acetonitrile, B=0.0375% TFA in H2O) or (b) aflow rate of 1.0 ml/min, using a solvent system of 90% A+10% B to 10%A+80% B over 2 minutes, where A=0.05% formic acid in water, B=0.05%formic acid in ACN;

(viii) X-ray powder diffraction spectra were determined (using aP'Analytical Cubix Analytical Instrument) by mounting a sample of thecrystalline material on a single silicon crystal (SSC) wafer sampleholder and spreading out the sample into a thin layer. The sample wasspun at 30 revolutions per minute (to improve counting statistics) andirradiated with X-rays generated by a copper long-fine focus tubeoperated at 45 kV and 40 mA with a wavelength of 1.5418 angstroms. Thecollimated X-ray source was passed through an automatic variabledivergence slit set at V20 and the reflected radiation directed througha 5.89 mm antiscatter slit and a 9.55 mm detector slit. The sample wasexposed for 100 seconds per 0.02° 2-theta increment (continuous scanmode) over the range 2 degrees to 40 degrees 2-theta in theta-thetamode. The instrument was equipped with a Position sensitive detector(Lynxeye). Control and data capture was by means of a Dell Optiplex 686Windows XP Workstation operating with X'Pert industry software. Personsskilled in the art of X-ray powder diffraction will realise that therelative intensity of peaks can be affected by, for example, grainsabove 30 microns in size and non-unitary aspect ratios that may affectanalysis of samples. The skilled person will also realise that theposition of reflections can be affected by the precise height at whichthe sample sits in the diffractometer and the zero calibration of thediffractometer. The surface planarity of the sample may also have asmall effect. Hence the diffraction pattern data presented are not to betaken as absolute values.

(ix) Differential Scanning Calorimetry was performed using a TAInstruments Q2000 DSC instrument. Typically less than 5 mg of materialcontained in a TZero aluminium pan fitted with a lid was heated over thetemperature range 0° C. to 300° C. at a constant heating rate of 10° C.per minute. A purge gas, nitrogen, was used —flow rate 50 ml per minute.Generally a measurement error of approximately plus or minus 1° C. inthe transition temperature values can be seen due to particle and samplesize introducing thermal lag; and

(x) the following abbreviations have been used:—

-   -   Boc₂O Di-tert-butyl dicarbonate    -   Br₂ bromine    -   CDCl₃ deuterated chloroform    -   DCM dichloromethane    -   DMAP 4-dimethylaminopyridine    -   DMF N,N-dimethylformamide    -   DMSO dimethyl sulphoxide    -   EtOAc ethyl acetate    -   HATU O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium        hexafluorophosphate    -   HCl hydrogen chloride    -   HF hydrogen fluoride    -   IPA isopropyl alcohol    -   KOAc potassium acetate    -   K₂CO₃ potassium carbonate    -   LiAlH₄ lithium aluminium hydride    -   MeCN acetonitrile    -   MeOD deutero-methanol    -   MgSO₄ magnesium sulphate    -   MsCl methanesulphonyl chloride    -   MTBE methyl tert-butyl ether    -   Na₂CO₃ sodium carbonate    -   NaOH sodium hydroxide    -   Na₂SO₄ sodium sulphate    -   NMP 1-methyl-2-pyrrolidone    -   N₂ nitrogen gas    -   PCy₃ tricyclohexylphosphine    -   Pd(dppf)Cl₂        dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium(II)    -   Pd—Cl₂ dichloropalladium(II)    -   PE petroleum ether    -   TBSCl tert-butyldimethylsilyl chloride    -   THF tetrahydrofuran    -   TEA triethyl amine    -   TLC thin layer chromatography

EXAMPLE 13-Amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidetri-hydrochloride

The title compound was prepared in accordance with the following scheme:

(i) 3-(4-Bromophenyl)propan-1-ol

To a solution of 3-(4-bromophenyl)-propionic acid (9.16 g, 40 mmol) inTHF (150 mL) at 0° C. was added LiAH₄ (1.4 g, 36 mmol) in portionwise.When the addition was completed, the reaction mixture was heated andstirred under reflux for additional 5 hours. The reaction mixture wascooled to 0° C. and 2 N—HCl (50 mL) added dropwise. The reaction mixturewas extracted with EtOAc and washed with water (×3). The combinedorganic extract was dried over anhydrous MgSO₄, filtered andconcentrated in vacuo, which afforded the subtitle compound (i) as anoil (8 g).

¹H-NMR (400 MHz, CDCl₃) δ 7.40 (d, 2H), 7.08 (d, 2H), 3.66 (t, 2H), 2.66(t, 2H), 1.89-1.82 (m, 2H).

(ii) (3-(4-Bromophenyl)propoxy)(tert-butyl)dimethylsilane

To a solution of the product of step (i) (32.25 g, 0.15 mol) inanhydrous dichloromethane (400 mL) was added triethylamine (45 g, 0.45mol), DMAP (915 mg, 7.5 mmol) and TBSCl (33 g, 0.215 mol) sequentially.The resulting solution was stirred overnight. The reaction mixture wasextracted with dichloromethane and washed with water (×3). The combinedorganic layer was dried over anhydrous MgSO₄, filtered and concentratedin vacuo. The crude product was purified by column chromatography withPE to afford the subtitle compound (ii) as a pale brown oil (40 g).

¹H-NMR (400 MHz, CDCl₃) δ 7.34 (d, 2H), 7.02 (d, 2H), 3.56 (t, 2H), 2.58(t, 2H), 1.77-1.73 (m, 2H), 0.86 (s, 9H), 0.00 (s, 6H);

HPLC Retention Time=1.017 min.

(iii) Methyl 3-amino-6-bromopyrazine-2-carboxylate

3-Aminopyrazine-2-carboxylic acid methyl ester (200 g, 1.3 mol, 3BScientific Corporation) was dissolved in AcOH (1 L). The solution waswarmed to 50° C. and Br₂ (312 g, 1.9 mol) added dropwise. When theaddition was completed, the mixture was stirred at 50° C. for additional3 hours. The reaction mixture was poured slowly to ice-water (4 L). Theprecipitate was filtered, washed with water and dried under a reducedpressure to afford the subtitle compound (iii) as a red brown solid (300g).

¹H-NMR (400 MHz, CDCl₃) δ 8.28 (s, 1H), 3.97 (s, 3H);

HPLC Retention Time=1.016 min.

(iv) 3-Amino-6-bromopyrazine-2-carboxylic acid

To a solution of the product of step (iii) (200 g, 0.86 mol) in MeOH(500 mL) was added 5 N—NaOH (500 mL) slowly. The resulting mixture wasstirred at 50° C. for 3 hours. MeOH was removed under a reduced pressureand water (300 mL) added to the reaction mixture. The solution wasacidified to pH 3 with 6N—HCl. The solution was extracted with EtOAc andwashed with water. The combined organic extract was dried over Na₂SO₄,filtered and evaporated to dryness to afford the subtitle compound (iv)as a brown yellow solid (180 g).

¹H-NMR (400 MHz, CDCl₃) δ 8.30 (s, 1H);

HPLC Retention Time=0.850 min.

(v) 3-Amino-6-bromo-N-(pyridin-3-yl)pyrazine-2-carboxamide

To a solution of the product of step (iv) (100 g, 0.45 mol) in DMF (350mL) was added TEA (129 mL, 0.91 mol) and 3-aminopyridine (42.3 g, 0.45mol). HATU (174 g, 0.45 mol) was added to the solution portionwise. Thesuspension was filtered, washed with water and dried under a reducedpressure to afford the subtitle compound (v) as a yellow solid (105 g).

¹H-NMR (400 MHz, CDCl₃) δ 9.50 (s, 1H), 8.81 (d, 1H), 8.40 (dd, 1H),8.32 (s, 1H), 8.23-8.20 (m, 1H), 7.33 (dd, 1H). LCMS (ESI) m/z[M+H]+=294&296 (calc=294&296) (MultiMode+), HPLC Retention Time=0.478min.

(vi)3-Amino-6-(4-(3-(tert-butyldimethylsilyloxy)propyl)phenyl)-N-(pyridin-3-yl)-pyrazine-2-carboxamide

A mixture of the product of step (ii) (9.84 g, 30 mmol),bis(pinacolato)diboron (11.4 g, 45 mmol), KOAc (9 g, 90 mmol) andPd(dppf)Cl₂ (1 g, 1.5 mmol) in dioxane (200 mL) was stirred and degassedwith N₂ (×3). The mixture was stirred at 80° C. under a N₂ atmospherefor 3 hours. The reaction mixture was cooled to room temperature. Water(25 mL) and Na₂CO₃ (10 g, 90 mmol) were added to the mixture. Themixture was stirred for 10 minutes before the product of step (v) (7.9g, 27 mmol) and Pd(dppf)Cl₂ (0.5 g, 0.7 mmol) were added. The resultingsolution was degassed again with N₂ (×3). The reaction mixture wasstirred at 90° C. under a N₂ atmosphere for additional 10 hours. Na₂SO₄was added, filtered and the filtrate concentrated in vacuo. The crudeproduct was purified by column chromatography with PE:EtOAc=5:1 to 3:1as eluent to afford the subtitle compound (vi) as a yellow solid (7.8g).

¹H-NMR (400 MHz, CDCl₃) δ 9.90 (s, 1H), 8.73 (d, 1H), 8.62 (s, 1H), 8.34(d, 1H), 8.21 (d, 1H), 7.74 (d, 2H), 7.28 (d, 2H), 7.28 (m, 1H), 3.6 (t,2H), 2.68 (t, 2H), 1.83-1.79 (m, 2H), 0.86 (s, 9H), 0.00 (s, 6H); LCMS(ESI) m/z [M+H]+=464 (calc=464) (MultiMode+),

HPLC Retention Time=1.233 min.

(vii)3-Amino-6-(4-(3-hydroxypropyl)phenyl)-N-(pyridin-3-yl)pyrazine-2-carboxamide

The product of step (vi) (14.5 g, 31.3 mmol) was added to a plasticvessel and THF (150 mL) added. HF (30 mL) was added carefully to thesolution and the solution stirred at room temperature for 3 hours. Themixture was poured to 600 mL of water and 5 N—NaOH added to basify thesolution to pH=10-11. The solution was extracted with EtOAc and washedwith water (×3). The combined EtOAc extract was dried over MgSO₄,filtered and concentrated in vacuo, which afforded the subtitle compound(vii) as a yellow solid (10 g).

¹H-NMR (400 MHz, DMSO-d₆) δ 8.94 (d, 1H), 8.87 (s, 1H), 8.31 (dd, 1H),8.18-8.16 (m, 1H), 8.10 (d, 2H), 7.60 (brs, 2H), 7.38 (dd, 1H), 7.28 (d,2H), 3.39 (q, 2H), 2.61 (t, 2H), 1.75-1.65 (m, 2H); LCMS (ESI) m/z[M+H]+=350 (calc=350) (MultiMode+), HPLC Retention Time=0.858 min.

(viii) 3-(4-(5-Amino-6-(pyridin-3-ylcarbamoyl)pyrazin-2-yl)phenyl)propylmethanesulfonate

Dichloromethane (200 mL) and diisopropylethylamine (2 g, 17.16 mmol)were added to the product of step (vii) (4 g, 11.46 mmol). The resultingsolution was cooled to 0° C. and MsCl (2 g, 17.16 mmol) added dropwise.When the addition was completed, the resulting solution was stirred at0° C. for 10 minutes and at room temperature for 2 hours. The reactionmixture was extracted with dichloromethane and washed with water (×3).The combined organic extract was dried over MgSO₄, filtered andconcentrated in vacuo, which afforded the subtitle compound (viii) as ayellow brown solid (4.5 g).

¹H-NMR (400 MHz, CDCl₃) δ 9.94 (s, 1H), 8.81 (d, 1H), 8.70 (s, 1H), 8.41(d, 1H), 8.30 (d, 1H), 7.84 (d, 2H), 7.34 (d, 2H), 7.34 (m, 1H), 4.27(t, 2H), 3.03 (s, 3H), 2.84 (t, 2H), 2.17-2.10 (m, 2H);

LCMS (ESI) m/z [M+H]+=428 (calc=428) (MultiMode+), HPLC RetentionTime=0.933 min.

(ix)3-Amino-6-(4-(3-(3-methoxypropylamino)propyl)phenyl)-N-(pyridin-3-yl)-pyrazine-2-carboxamidetri-hydrochloride

A mixture of the product of step (viii) (4.25 g, 10 mmol), K₂CO₃ (2.76g, 20 mmol) and 3-methoxypropylamine (1.78 g, 20 mmol) in MeCN (100 mL)was stirred at 90° C. under a N₂ atmosphere overnight. The reactionmixture was cooled to room temperature and filtered. The filtrate wasconcentrated in vacuo and purified by a preparative HPLC. The fractionscontaining the desired compound were evaporated to reduce the volume ofthe solvent (acetonitrile and water) from 1 L to 50 ml and 0.5 M-aqueousHCl (20 mL) added.

The resulting yellow solution was then lyophilized, which afforded thetitle compound as a tri-hydrochloride salt (yellow solid, 2.7 g).

¹H-NMR (400 MHz, MeOD) δ 9.63 (d, 1H), 8.95 (d, 1H), 8.81 (s, 1H), 8.64(d, 1H), 8.12-8.09 (m, 1H), 8.10 (d, 2H), 7.42 (d, 2H), 3.51 (t, 2H),3.34 (s, 3H), 3.13 (t, 2H), 3.05 (t, 2H), 2.78 (t, 2H), 2.10-2.00 (m,2H), 1.98-1.90 (m, 2H);

LCMS (ESI) m/z [M+H]+=421 (calc=421) (MultiMode+), HPLC RetentionTime=0.815 min.

EXAMPLE 23-Amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-carboxamide

The title compound was prepared in accordance with the following scheme:

(i) 3-(4-Bromophenyl)propyl methanesulphonate

To a stirred solution of 3-(4-Bromophenyl)propan-1-ol (500 g, 2.30 mol)and diisopropylethylamine (312 g, 2.40 mol) in anhydrous dichloromethane(4 L) at 0˜5° C. was added dropwise methanesulphonyl chloride (280 g,2.40 mol) slowly. After addition, the reaction mixture was stirred for 1hour at 0˜5° C. The reaction mixture was washed with brine (1 L) twice.The organic phase was concentrated in vacuo to afford the subtitlecompound (i) (667 g). HPLC Retention Time=1.353 min.

(ii) 3-(4-Bromophenyl)-N-(3-methoxypropyl)propan-1-amine

A mixture of the product of step (i) (272 g, 0.93 mol), K₂CO₃ (256 g,1.86 mol), methoxypropylamine (199 g, 2.22 mol) in MeCN (1.5 L) wasstirred at 80-90° C. under N₂ atmosphere for 16 hour. The reactionmixture was cooled to room temperature and filtered.

The filtrate was concentrated to remove MeCN. MTBE (1 L) and H₂O (1 L)were charged to the resulting mixture and washed with aqueous 1N—HCl (1L). The aqueous phase was collected and basified by aqueous 2.5N—NaOH topH=9-10, to afford the subtitle compound (ii) in solution, which wasused for next step directly.

LCMS (ESI) m/z [M+H]+=286&288 (calc=286&288) (MultiMode+), HPLCRetention Time=0.935 min.

(iii) tert-ButylN-[3-(4-bromophenyl)propyl]-N-(3-methoxypropyl)carbamate

To a stirred reaction mixture of the product of step (ii) (0.93 mol) inaqueous NaOH solution from last step, was charged with THF (1 L) andBoc₂O (222 g, 1.01 mol) added to the mixture dropwise at 10° C. Afteraddition, the mixture was stirred for 1 hour at 10° C. and extractedwith MTBE (1 L×2). The organic phase was concentrated and purified bycolumn chromatography (PE:EtOAc=10:1-5:1) to afford 300 g of thesubtitle compound (iii). ¹H NMR (400 MHz, CDCl₃) δ 7.38 (d, J=8.4 Hz,2H), 7.05 (d, J=8.4 Hz, 2H) 3.35 (t, J=6.0 Hz, 2H), 3.30 (s, 3H), 3.22(s, 4H), 2.54 (t, J=7.6 Hz, 2H), 1.76-1.84 (m, 4H), 1.43 (s, 9H). LCMS(ESI) m/z [M+H]+=386&388 (calc=386&388) (MultiMode+).

(iv) tert-ButylN-(3-methoxypropyl)-N-{3-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]propyl}carbamate

A mixture of the product of step (iii) (230 g, 0.60 mol),bis(pinacolato)diboron (227 g, 0.89 mol), KOAc (175 g, 1.79 mol), Pd—Cl₂(3.20 g, 18 mmol), PCy₃ (10 g, 38 mmol) in MeCN (4 L) was stirred anddegassed with N₂ three times. The reaction mixture was stirred at 80° C.under N₂ for 16 hours. The mixture was filtered and the filtrateconcentrated in vacuo to remove MeCN. MTBE (3 L) was charged and theorganic phase washed with H₂O (800 mL×3) and concentrated to obtain thesubtitle compound (iv) as an oil (250 g), which was used for next stepdirectly. ¹H-NMR (400 MHz, CDCl₃) δ 7.71 (d, J=8.0 Hz, 2H), 7.18 (d,J=8.0 Hz, 2H), 3.35 (t, J=6.0 Hz, 2H), 3.29 (s, 3H), 3.22 (s, 4H), 2.60(t, J=7.6 Hz, 2H), 1.75-1.88 (m, 4H), 1.43 (s, 9H), 1.32 HPLC RetentionTime=1.793 min.

(v) tert-ButylN-[3-{4-[5-amino-6-(3-pyridylcarbamoyl)pyrazin-2-yl]phenyl}propyl]-N-(3-methoxypropyl)carbamate

A mixture of the product of step (iv) (250 g, 0.58 mol),3-amino-6-bromo-N-(pyridin-3-yl)pyrazine-2-carboxamide (220 g, 0.75mol), Na₂CO₃ (183 g, 1.73 mol) and Pd(dppf)Cl₂ (25.30 g, 35 mmol) in DMF(3 L) was stirred and degassed with N₂ three times. The mixture wasstirred at 70˜80° C. under N₂ for 24 hours. The mixture was filtered andthe filtrate concentrated to in vacuo to remove DMF. EtOAc (1.5 L) andH₂O (800 mL) were charged and stirred for 1 hour at 10-25° C. Thesuspension was filtered, the organic phase was concentrated in vacuo andpurified by column chromatography (PE: EtOAc=3:1-1:3). The crude productwas re-crystallized from EtOAc-MeOH and filtered, the yellow solid waswashed by EtOA and dried under reduced pressure. The subtitle compound(v) was obtained (pale yellow solid, 160 g).

LCMS (ESI) m/z [M+H]+=521 (calc=521) (MultiMode+), HPLC RetentionTime=1.366 min.

(vi)3-Amino-6-(4-(3-(3-methoxypropylamino)propyl)phenyl)-N-(pyridin-3-yl)-pyrazine-2-carboxamide

A mixture of the product of step (v) (300 g, 0.58 mol) in 4N—HCl inEtOAc (3 L) was stirred for 3 hours. The suspension was filtered and theyellow solid obtained. The solid was dissolved in H₂O (1.2 L). 2N—NaOHwas added dropwise into the solution to basified to pH=11. The mixturewas extracted with a mixture of THF and EtOAc (5 L×2, 1:1) and theorganic phase washed with aqueous 10%—NaCl and concentrated in vacuo.The subtitle compound (vi) was obtained (pale yellow solid, 210 g).

¹H NMR (400 MHz, DMSO-d₆) δ 10.57 (s, 1H), 8.99 (d, J=2.4 Hz, 1H), 8.89(s, 1H), 8.35 (dd, J=1.2 Hz, J=4.8 Hz, 1H), 8.23-8.20 (m, 1H), 8.12 (d,J=8.4 Hz, 2H), 7.67 (s, 2H), 7.40 (dd, J=4.8 Hz, J=4.8 Hz, 1H), 7.28 (d,J=8.4 Hz, 2H), 3.33 (t, J=6.8 Hz, 2H), 3.18 (s, 3H), 2.62 (t, J=7.2 Hz,2H), 2.47 (dd, J=7.2 Hz, J=14 Hz, 4H), 1.73-1.65 (m, 2H), 1.62-1.56 (m,2H).

The3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamideForm B prepared as described above was determined to be crystalline byXRPD (see FIG. 4) and had the following X-Ray Powder Diffraction peaks:

Angle 2- Theta (2θ) Intensity % 3.5 100 7.0 4 9.5 2 10.4 15 12.4 3 13.82 14.1 8 15.9 7 17.6 9 21.0 5

DSC analysis of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamideForm B was also carried out (FIG. 5), using a heating rate of 10°C./min, and showed an initial endothermic event with an onset at 47° C.and a peak at 60° C. followed by a subsequent phase transition with anonset of 113° C. and a peak at 116° C. Thus, DSC analysis shows that3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamideForm B is a high melting solid with an onset of melting at about 113° C.and a peak at about 116° C.

EXAMPLE 3A Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate

500.6 mg of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamide(as prepared in Example 2) and 189.09 mg of Benzenesulfonic acid wereaccurately weighed into a glass vial and the 15 mL IPA was added to forma suspension. The suspension was kept shaking for 2 days at roomtemperature, and then filtered and dried at room temperature to yield ayellow powder.

The Form A3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate prepared as described above was determined to becrystalline by XRPD (see FIG. 6 a) and had the following X-Ray Powder

Diffraction Peaks:

Angle 2- Theta (2θ) Intensity % 7.5 16 8.4 100 11.0 13 14.5 9 15.7 1218.5 8 20.2 6 22.0 11 22.2 10 23.0 3

DSC analysis of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate Form A was also carried out, using a heating rate of 10°C./min, and showed an initial endothermic event with an onset at 89° C.and a peak at 99° C. followed by a melt with an onset of 164° C. and apeak at 167.0° C. (FIG. 7 a). Thus, DSC analysis showed that3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate Form A is a high melting solid with an onset of melting atabout 164° C. and a peak at about 167° C.

EXAMPLE 3B Form C of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate

Synthesis of 3-Amino-6-bromo-N-(pyridin-3-yl)pyrazine-2-carboxamideStage 1: Methyl 3-amino-6-bromopyrazine-2-carboxylate

3-Aminopyrazine-2-carboxylic acid methyl ester (1 kg),N-bromosuccinimide (1.162 kg and acetonitrile (8 L) were charged intoreactor. The mixture was stirred at room temperature for 10 min, heatedto 75-80° C. and held for 2-3 hours under slightly refluxing conditions.The mixture was cooled to 25° C. and evaporated to dryness on a rotaryevaporator. The resulting dark solids were redissolved indichloromethane (20 L) and activated carbon (100 g) was charged. Theresulting suspension was stirred at 25° C. for 30-60 minutes beforefiltering to remove insolubles. The filtrate was subsequently washedwith saturated Na₂SO₃ (3×5 L) aqueous solution and water (5 L). TheOrganic layers were collected after phase separation. Dichloromethanewas removed on rotary evaporator and Methyl3-amino-6-bromopyrazine-2-carboxylate was obtained as a brown solidwhich was used in the next step without further purification (wetproduct 1090 g).

Stage 2: 3-amino-6-bromopyrazine-2-carboxylic acid

Methyl 3-amino-6-bromopyrazine-2-carboxylate (1.4 Kg), 5N NaOH solution(5.6 L) and methanol (8.4 L) were charged into reactor. The resultingsuspension was warmed at 50° C. for 3 hours with vigorous stirring. Themixture was analysed for reaction completion. The mixture was cooled to25° C. and the solids were collected by filtration. Filter cake waswashed with water (1 L). The resultant solids were suspended in water (5L) and pH value of this suspension was adjusted to 3 by adding 6 N HClsolution. The acidic solution was vigorously stirred at 25° C. for 48 h.During this period, pH value was checked, additional acid was charged ifpH value >3. The suspension was filtered to collect a yellow-brownsolid, which was dried in oven at 40° C. under a flow of N₂. The crudesubtitle material was used in the next step.

Stage 3: 3-Amino-6-bromo-N-(pyridin-3-yl)pyrazine-2-carboxamide

3-Amino-6-bromopyrazine-2-carboxylic acid (1.07 kg), 3-aminopyridine(460 g), triethylamine (680 ml) and DMF (6.4 L) were charged into areactor. The mixture was cooled to −5 to 0° C. and stirred for 15 min.Under a nitrogen flow, HATU (1.8 kg) was slowly added in many portionswhile maintaining the temperature below 10° C. Upon addition, thickyellow precipitate formed. After addition, the mixture was stirred at20-25° C. for 1 h. The reaction was analysed for completion(3-Amino-6-bromopyrazine-2-carboxylic acid=0%). Water (13 L) was addedunder vigorous stirring. The yellow suspension was stirred at 25° C. for30 min. The suspension was filtered and the filter cake was rechargedinto reactor. Water (13 L) was charged. The yellow suspension was warmedto 50° C. and stirred at this temperature for 2 h. The suspension wasfiltered and the filter cake was washed with water. The wet product(3-Amino-6-bromo-N-(pyridin-3-yl)pyrazine-2-carboxamide) was dried inoven at 55° C. under a flow of nitrogen. (1.14 Kg). ¹H-NMR (400 MHz,DMSO-d₆) 7.39 (dd, 1H); 7.74 (br.s., 2H); 8.18 (ddd, 1H); 8.33 (dd, 1H);8.43 (s, 1H); 8.96 (d, 1H); 10.51 (s, 1H).

Synthesis of tert-butyl(3-methoxypropyl){3-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]propyl}carbamateStage 1: 3-(4-bromophenyl)propan-1-ol

Sodium borohydride (119.12 g) and THF (5 L) were charged into reactor 1and temperature adjusted to −20° C. 3-(bromophenyl)propionic acid (1 kg)was charged into reactor 2 with THF (3 L) and stirred at 25° C. untilall the solids dissolved. This solution in reactor 2 was transferred toreactor 1 maintaining reactor 1 contents at <0° C. BF₃.Et₂O (991.4 g)was slowly added into reactor 1 maintaining reactor 1 contents at −20 to−10° C. After addition, the mixture was stirred at −20 to −10° C. for 1h. The reaction is analysed for completion (3-(bromophenyl)propionicacid <2.5%). Water (2 L) was slowly added into reactor 1 to quench thereaction. (gas evolution). MTBE (5 L) was charged into reactor 1 andphases separation was carried out. The upper layer was collected andlower layer was washed with MTBE (5 L). The upper layer was collectedand combined with the layer from the previous step. Adjusted pH value ofthe organic layer to 8-9 by adding 1N NaOH solution. The organic layerwas washed with saturated NaCl solution (2 L) and the upper layer wascollected. The organic layer was evaporated down to a colourless oil.

Stage 2: 3-(4-bromophenyl)propyl methanesulfonate

3-(4-Bromophenyl)propan-1-ol (335 g) and dichloromethane (2.5 L) wascharged into Reactor 1. Reactor 1 was adjusted to −10 to 0° C.Triethylamine (205 g) was charged into Reactor 1. Methanesulphonylchloride (272.7 g) was added dropwise into Reactor 1 maintainingcontents at <0° C. Reactor 1 was stirred at −5 to 5° C. for 1 h thenanalysed for reaction completion. The organic layer was washed withwater (1 L) and saturated NaCl solution (1 L). The organic layer wasevaporated down to dryness to give 3-(4-bromophenyl)propylmethanesulfonate (418 g).

Stage 3: 3-(4-bromophenyl)-N-(3-methoxypropyl)propan-1-amine

3-(4-bromophenyl)propyl methanesulfonate (418 g) and acetonitrile (2 L)were charged into reactor 1. 3-methoxypropylamine (305 g) and potassiumcarbonate (394 g) were added to reactor 1 and the mixture was warmed to80-90° C. for 4-16 h. The mixture was analysed for completion(3-(4-bromophenyl)propyl methanesulfonate <0.5%). The reaction wascooled to 25° C. and the suspension was filtered to remove the whitesolids. The filtrate was concentrated to dryness on a rotary evaporator.The crude product from the rotary evaporator, MTBE (2 L) and water (1.5L) were charged into reactor 2. After phase separation, the organiclayer was collected and washed with 1N HCl (2 L) until pH=4-5. Afterphase separation, the aqueous phase was basified by 2.5N NaOH solutionto pH=9-10 and the resulting aqueous solution was used in the next stepwithout further treatment.

Stage 4: tert-butyl[3-(4-bromophenyl)propyl](3-methoxypropyl)carbamate

3-(4-bromophenyl)-N-(3-methoxypropyl)propan-1-amine (500 g) in water(1.5 L) and THF (500 ml) was charged into reactor Triethylamine (176.8g) was charged into reactor at 10° C. Boc₂O (475.5 g) was added dropwiseto the mixture in reactor 1 at 10° C. and the mixture was stirred for 1h at 10° C. The mixture was analysed for reaction completion. Themixture was extracted with MTBE (3×1 L). The combined organic layerswere evaporated to down to give crude product as oil. The crude productwas further purified by silica gel column chromatography. (Petroleumether:Ethyl acetate=1:0-20:1-10:1).tert-butyl[3-(4-bromophenyl)propyl](3-methoxypropyl)carbamate wasobtained as slightly yellow oil. (327.0 g)

Stage 5: tert-butyl (3-methoxypropyl){3-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]propyl}carbamate

tert-butyl[3-(4-bromophenyl)propyl](3-methoxypropyl)carbamate (500 g)and acetonitrile (4.3 L) were charged into reactor 1 along withbis(pinacolato)diboron (394.4 g), potassium acetate (190.5 g), PCy₃(23.2 g) and Pd(OAc)₂ (8.7 g). The vessel was swing purged with nitrogenthree times. The mixture was warmed to 85-90° C. for 16 h then analysedfor complete reaction(tert-butyl[3-(4-bromophenyl)propyl](3-methoxypropyl)carbamate <0.5%).The mixture was cooled to 25° C., filtered and the filtrate wasconcentrated to remove MeCN. MTBE (3 L) was charged and MTBE layerwashed with water (3×1.5 L). The MTBE layer was concentrated to givecrude product. The crude product was further purified by silica gelcolumn chromatography. (Petroleum ether:Ethyl acetate=1:0-40:1-10:1).tert-butyl (3-methoxypropyl){3-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]propyl}carbamatewas obtained as slightly yellow oil. (490.0 g) ¹H-NMR (400 MHz, DMSO-d₆)δ ppm 1.27 (s, 12H); 1.35 (m, 9H); 1.65 (m, 2H); 1.74 (m, 2H); 2.54 (t,2H); 3.10 (m, 1H); 3.15 (m, 1H); 3.18 (s, 3H); 3.26 (t, 2H); 7.19 (d,2H); 7.58 (d, 2H).

Synthesis of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate Stage 1: (tert-butyl(3-{4-[5-amino-6-(pyridin-3-ylcarbamoyl)pyrazin-2-yl]phenyl}propyl)(3-methoxypropyl)carbamate

To a reactor was charged,3-Amino-6-bromo-N-(pyridin-3-yl)pyrazine-2-carboxamide (1580 g),tert-butyl (3-methoxypropyl){3-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]propyl}carbamate(2778 g) 1-butanol (23.7 L), potassium phosphate, tribasic, N-hydrate(2740 g) and water (1.58 L). The contents were stirred under nitrogen atambient (21° C.). In a second reactor was made up a solution of3-(di-tert-butylphosphonium) propane sulphonate (DTBPPS) (83.04 g);sodium tetrachloropalladate (45.52 g) and water (1.58 L). Thecatalyst/ligand solution was poured into the reaction; the reaction wasinerted and heated up to 80° C. The reaction was stirred for 2 hours at80° C. The batch was cooled to 60° C. and water (22.1 L) was added. Thecontents washed with water at 60° C. The organic layer and interfacewere filtered through perlite (500 g) made up with 1-Butanol (3.95 L).The organic liquors were recharged to the vessel and washed with water(11.1 L) at 60° C. The organic liquor was washed with 10% w/v aqueoussodium chloride (11.2 L) at 60° C. The organic liquors were heated up to60° C. and distilled under vacuum to ˜10 relative volumes. The batch wascooled to 20° C. and left to stir for 16 hours under nitrogen. Thecontents were filtered and washed with 1-Butanol (4 L×2). The yellowfree flowing solid was discharged and dried in vacuum oven at 40° C.overnight (tert-butyl(3-{4-[5-amino-6-(pyridin-3-ylcarbamoyl)pyrazin-2-yl]phenyl}propyl)(3-methoxypropyl)carbamate(2428 g)).

¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.37 (s, 9H) 1.62-1.72 (m, 2H) 1.73-1.87(m, 2H) 2.59 (t, J=7.65 Hz, 2H) 3.12-3.21 (m, 4H) 3.19 (s, 3H) 3.28 (t,J=6.25 Hz, 2H) 7.31 (d, J=8.10 Hz, 2H) 7.42 (dd, J=8.41, 4.74 Hz, 1H)7.62 (br.s., 2H) 8.13 (d, J=8.10 Hz, 2H) 8.21 (ddd, J=8.40, 2.60, 1.40Hz, 1H) 8.35 (dd, J=4.70, 1.40 Hz, 1H) 8.90 (s, 1H) 8.98 (d, J=2.59 Hz,1H) 10.54 (s, 1H)

Stage 2:3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamide

Tert-butyl(3-{4-[5-amino-6-(pyridin-3-ylcarbamoyl)pyrazin-2-yl]phenyl}propyl)(3-methoxypropyl)carbamate(1 kg) and water (8 L) were added to the reactor, stirred and heated upto 70° C. 36% Hydrochloric acid (606 ml) was added slowly controllingrate of gas evolution. The contents were stirred for 45 minutes at 70°C. The pH was adjusted to 3-4 by addition of sodium hydroxide (325 ml).The contents were cooled to 50° C. and 2-methyltetrahydrofuran (5 L) wasadded. The pH was then adjusted to pH 11-12 by the addition of 48%sodium hydroxide liquor (170 ml). The contents were then cooled to 40°C. The batch allowed to settle and the two layers separated. The aqueouslayer was washed with 2-methyltetrahydrofuran (2.5 L). The combinedorganic layers were washed with a solution of 10% w/v aqueous sodiumchloride solution. The organic layer was distilled to remove 3.75 L ofdistillate. The remaining liquors were heated to 50° C. andmethyl-t-butyl ether (4 L) added. The liquors were cooled to 10° C. over4 hours then isolated by filtration. The solid was washed withmethyl-t-butyl ether (2 L×2) and dried in a vacuum oven at 50° C.3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamide(721 g).

¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.77-1.87 (m, 2H) 1.88-2.01 (m, 2H) 2.72(t, J=7.65 Hz, 2H) 2.89-3.03 (m, 4H) 3.23 (s, 3H) 3.39 (t, J=5.98 Hz,2H) 7.35 (d, J=8.30 Hz, 2H) 7.68 (br.s., 2H) 7.68 (dd, J=8.62, 5.17 Hz,1H) 8.18 (d, J=8.30 Hz, 2H) 8.41-8.52 (m, 2H) 8.57 (br.s., 2H) 8.94 (s,1H) 9.16 (d, J=2.15 Hz, 1H) 10.77 (s, 1H)

Stage 3:3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate

To reactor was charged3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamide(700 g) and ethanol (4.9 L). The contents were heated up to 60° C. togive a solution. Benzene sulphonic acid (258 g) was charged to thereactor followed by ethanol (2.1 L) and water (70 ml). The contentsheated up to 60° C. The complete solution was then cooled to 51° C. andseeded with Form A of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate (0.35 g, as prepared in accordance with Example 3a). Oncecrystallisation was established the contents were held at 51° C. for 2hours then ramp cooled to 40° C. over 4 hours, held at 40° C. for 4hours then further cooled to 10° C. over 4 hours. The contents were heldat 10° C. for 12 hours then filtered and washed with ethanol (700 ml×2).The solid3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate Form C was dried in a vacuum oven at 40° C. (810 g).¹H-NMR (700 MHz, DMSO-d₆) δ ppm 1.78-1.86 (m, 2H) 1.92 (quin, J=7.70 Hz,2H) 2.70 (t, J=7.70 Hz, 2H) 2.89-2.99 (m, 4H) 3.22 (s, 3H) 3.37 (t,J=6.05 Hz, 2H) 7.28-7.35 (m, 5H) 7.47 (dd, J=8.25, 4.73 Hz, 1H) 7.62(br.s., 2H) 7.62 (dd, J=7.92, 1.54 Hz, 2H) 8.16 (d, J=8.36 Hz, 2H) 8.27(ddd, J=8.30, 2.40, 1.30 Hz, 1H) 8.34 (br.s., 2H) 8.38 (dd, J=4.70, 1.32Hz, 1H) 8.91 (s, 1H) 9.01 (d, J=2.42 Hz, 1H) 10.57 (s, 1H)

The Form C3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate prepared as described directly above was determined to becrystalline by XRPD (see FIG. 6 b) and had the following X-Ray

Powder Diffraction Peaks:

Angle 2- Theta (2θ) Intensity % 11.2 0.9 12.6 0.3 14.7 0.6 15.5 0.7 15.80.7 16.8 0.8 20.4 0.7 21.1 4.3 22.5 1.8 26.5 0.3

DSC analysis of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate Form C was also carried out, using a heating rate of 10°C./min and showed a solid to solid transition with an onset of 98° C.and a peak at 99° C. followed by a subsequent phase transition with anonset of 165° C. and a peak at 170° C. (FIG. 7 b). Thus, DSC analysisshowed that3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-besylate Form C is a high melting solid with an onset of melting atabout 165° C. and a peak at about 170° C.

EXAMPLE 4.1-4.4 Particular salts of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamideEXAMPLE 4.1

3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-tosylate

EXAMPLE 4.2

3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidedi-esylate

EXAMPLE 4.3

3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-DL-mandelate

EXAMPLE 4.4

3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-benzoate

The mono-benzoate Form A, mono-DL-mandelate Form A and mono-tosylateForm A salts were prepared by reacting approximately 500 mg3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamideForm B with 1 molar equivalent of the appropriate counterion in 10 mL ofIPA. The reaction was carried out at room temperature and the mixturestirred for 5 days prior to isolation by filtration.

The di-esylate Form A was prepared by reacting approximately 500 mg3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamideForm B with 2 molar equivalents of counter-ion in 10 mL of IPA. Thereaction was carried out at room temperature and the mixture was stirredfor 5 days prior to isolation by filtration.

The3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-tosylate Form A was determined to be crystalline by XRPD (see FIG.8) and had the following X-Ray Powder Diffraction peaks:

Angle 2- Theta (2θ) Intensity % 8.0 100 11.7 19 14.8 24 20.0 10 20.9 1021.5 12 22.2 27 23.0 53 27.5 13 28.0 11

DSC analysis of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-tosylate Form A was also carried out (FIG. 9), using a heating rateof 10° C./min, and showed a melt with an onset of 168° C. and a peak at171° C. Thus, DSC analysis shows3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-tosylate Form A is a high melting solid with an onset of melting atabout 168° C. and a peak at about 171° C.

The3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidedi-esylate Form A was determined to be crystalline by XRPD (see FIG. 10)and had the following X-Ray Powder Diffraction peaks:

Angle 2- Theta (2θ) Intensity % 5.2 100 10.4 9 12.7 32 15.0 7 16.9 1517.3 13 19.1 21 19.6 11 20.0 5 23.5 15

DSC analysis of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidedi-esylate Form A was also carried out (FIG. 11), using a heating rateof 10° C./min, and showed a melt with an onset of 179° C. and a peak at181° C. Thus, DSC analysis shows that3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidedi-esylate Form A is a high melting solid with an onset of melting atabout 179° C. and a peak at about 181° C.

The3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-DL-mandelate Form A was determined to be crystalline by XRPD (seeFIG. 12) and had the following X-Ray Powder Diffraction peaks:

Angle 2- Theta (2θ) Intensity % 2.7 84 5.2 39 7.8 74 9.6 50 10.5 10012.3 13 13.1 41 15.7 20 18.4 51 2.7 84

DSC analysis of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-DL-mandelate Form A was also carried out (FIG. 13), using a heatingrate of 10° C./min, and showed an initial endothermic event thought tocorrespond to a melt, with an onset of about 140° C. and a peak at about144° C. This was followed by an exothermic (recrystallisation) eventwith an onset of about 146° C. and peak of about 149° C., followed by afinal meting event with an onset of about 153° C. and a peak of about156° C. Thus, DSC analysis shows that3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-DL-mandelate Form A undergoes a melt-recrystallisation-meltsequence of transitions with an initial an onset of melting at about140° C. and a peak at about 144° C.

The3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-benzoate Form A was determined to be crystalline by XRPD (see FIG.14) and had the following X-Ray Powder Diffraction peaks:

Angle 2- Theta (2θ) Intensity % 5.3 100 6.2 18 9.2 57 9.8 19 11.9 1416.6 25 17.8 38 18.1 56 19.5 20 24.6 39

DSC analysis of3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-benzoate Form A was also carried out (FIG. 15), using a heatingrate of 10° C./min, and showed a melt with an onset of 141° C. and apeak at 143° C. Thus, DSC analysis shows that3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidemono-benzoate Form A is a high melting solid with an onset of melting atabout 141° C. and a peak at 143° C.

EXAMPLE 53-Amino-6-(4-{3-[(2-methoxyethyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidetri-hydrochloride

The title compound was prepared using analogous methods to thosedescribed in Example 1, except that 2-methoxyethylamine was used insteadof 3-methoxypropylamine in step (ix).

¹H NMR (400 MHz, CD₃OD) δ 9.65 (s, 1H), 8.96 (d, J=8.0 Hz, 1H), 8.83 (s,1H), 8.64 (d, J=5.6 Hz, 1H), 8.12 (m, 3H), 7.42 (d, J=7.6 Hz, 2H), 3.66(m, 2H), 3.48 (s, 3H), 3.23 (m, 2H), 3.10 (m, 2H), 2.82 (m, 2H), 2.11(m, 2H).

LCMS: Retention Time=0.761 min.

EXAMPLE 63-Amino-6-(4-{2-[(3-methoxypropyl)amino]ethyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamide

The title compound was prepared in accordance with the followingprocedure:

(i) (4-Bromophenylethoxy)(tert-butyl)dimethylsilane

To a solution of 2-(4-bromophenyl)ethanol (5 g, 25 mmol) in anhydrousdichloromethane (150 mL) was added triethylamine (7.5 g, 75 mmol), DMAP(300 mg, 2.5 mmol) and tert-butyldimethylsilyl chloride (7.5 g, 50 mmol)sequentially. The resulting pale brown solution was stirred at roomtemperature for overnight. The reaction mixture was extracted withdichloromethane and water three times. The combined organic layers weredried over anhydrous sodiumsulphate, filtered and concentrated in vacuo.The crude product was subjected to column chromatography with PE aseluant to afford the subtitle compound as a brown oil (7.1 g).

¹H NMR (400 MHz, CDCl₃) δ 7.41 (dd, J=6.4, 2.0 Hz, 2H), 7.10 (dd, J=6.4,2.0 Hz, 2H), 3.80 (t, J=6.8 Hz, 2H), 2.79 (t, J=6.8 Hz, 2H), 0.88 (s,9H), 0.00 (s, 6H).

(ii)3-Amino-6-(4-(2-(tert-butyldimethylsilyloxy)ethyl)phenyl)-N-(pyridin-3-yl)pyrazine-2-carboxamide

A mixture of the product of step (i) (6.9 g, 27.1 mmol), KOAc (6.6 g,67.8 mmol), Pd(dppf)Cl₂ (0.82 g, 1.13 mmol) in dioxane (150 mL) wasdegassed and backfilled with N₂ three times. The mixture was stirred at90° C. under N₂ overnight. The reaction mixture was cooled to roomtemperature and the product of step (v) in Example 1 (6.6 g, 22.6 mmol),Pd(dppf)Cl₂ (0.5 g, 0.7 mmol), water (50 mL) and Na₂CO₃ (7.2 g, 67.8mmol) were added into the mixture. The resulting solution was degassedwith N₂ three times and stirred at 90° C. under N₂ for an additional 7hours. The reaction mixture was extracted with dichloromethane threetimes and the combined organic layers were dried over anhydroussodiumsulphate, filtered and concentrated in vacuo. The residue waspurified by column with PE:EtOAc=5:1→3:1 as eluants to afford thesubtitle compound as a yellow solid (4.1 g)

¹H NMR (400 MHz, CDCl₃) δ 9.95 (s, 1H), 8.79 (d, J=2.4 Hz, 1H), 8.68 (s,1H), 8.41 (d, J=2.4 Hz, 1H), 8.25 (d, J=8.2 Hz, 1H), 7.80 (d, J=8.0 Hz,2H), 7.39-7.30 (m, 3H), 3.84 (t, J=6.8 Hz, 2H), 2.88 (t, J=6.8 Hz, 2H),0.87 (s, 9H), 0.00 (s, 6H).

(iii)3-Amino-6-(4-(2-hydroxyethyl)phenyl)-N-(pyridin-3-yl)pyrazine-2-carboxamide

A solution of the product of step (ii) (4.1 g, 9.13 mmol) and tetrabutylammonium fluoride (4.8 g, 18.3 mmol) in THF (150 mL) was stirred at roomtemperature overnight. The mixture was concentrated in vacuo and theresidue extracted with dichloromethane and washed with water. Thecombined organic layers were dried over anhydrous sodium sulphate,filtered and concentrated in vacuo to obtain the subtitle compounds (2.5g).

LCMS Retention time=0.801 min

(iv) 4-(5-Amino-6-(pyridin-3-ylcarbamoyl)pyrazin-2-yl)phenethylmethanesulfonate

To a solution of the product of step (iii) (2.5 g, 7.5 mmol, 1.0 eq) andTEA (2.3 g, 22.5 mmol) in dry dichlomethane was added MsCl (1.7 g, 15mmol). The reaction mixture was stirred at room temperature for 4 hours.The solvent was removed under reduced pressure and the residu purifiedby column to afford the subtitled compounds (1.9 g).

¹H NMR (400 MHz, CDCl₃) δ 9.87 (s, 1H), 8.76 (s, 1H), 8.64 (s, 1H), 8.36(d, J=4.0 Hz, 1H), 8.24 (d, J=8.4 Hz, 1H), 7.80 (d, J=8.0 Hz, 2H),7.38-7.24 (m, 3H), 4.42 (t, J=6.8 Hz, 2H), 3.08 (t, J=6.8 Hz, 2H), 2.87(s, 3H).

(v)3-Amino-6-(4-{2-[(3-methoxypropyl)amino]ethyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamide

A mixture of the product of step (iv) (250 mg, 0.61 mmol), K₂CO₃ (250mg, 1.8 mmol), 3-methoxypropylamine (82 mg, 0.92 mmol) in MeCN (20 mL,Aldrich, HPLC grade) was stirred at 90° C. under N₂ atmosphereovernight. The reaction mixture was cooled to room temperature andfiltered. The filtrate was concentrated in vacuo and the residuesubjected to prep-HPLC for purification, the eluents were concentratedand 1N NaOH solution added into the residue to control the pH=8-9. Themixture was extracted with DCM three times. The combined extracts wasdried over anhydrous sodium sulphate, filtered, concentrated in vacuo,which afforded the title compound (yellow solid, 158 mg)

¹H NMR (400 MHz, CDCl₃) δ 9.89 (s, 1H), 8.74 (d, J=2.4 Hz, 1H), 8.63 (s,1H), 8.35 (d, J=4.4 Hz, 1H), 8.23 (d, J=8.4 Hz, 1H), 7.76 (d, J=8.4 Hz,2H), 7.40-7.27 (m, 3H), 3.37 (t, J=6.2 Hz, 2H), 3.23 (s, 3H), 2.90-2.77(m, 4H), 2.69 (t, J=7.0 Hz, 2H), 1.76-1.62 (m, 2H).

LCMS: Retention Time=0.741 min.

EXAMPLE 73-Amino-6-(4-{2-[(2-methoxyethyl)amino]ethyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidetri-hydrochloride

3-Amino-6-(4-{2-[(2-methoxyethyl)amino]ethyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamidewas prepared using analogous procedures to those described in Example 6,except that 2-methoxyethylamine was used instead of 3-methoxypropylaminein step (v). MeCN was added to3-Amino-6-(4-{2-[(2-methoxyethyl)amino]ethyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamideand 0.5 M-aqueous HCl (20 mL) was also added. The resulting yellowsolution was then lyophilized, which afforded the title compound as atri-hydrochloride salt.

¹H NMR (400 MHz, CD₃OD) δ 9.66 (s, 1H), 8.98 (d, J=8.8 Hz, 1H), 8.85 (s,1H), 8.65 (d, J=5.6 Hz, 1H), 8.17-8.12 (m, 3H), 7.48 (d, J=8.4 Hz, 2H),3.70 (t, J=4.8 Hz, 2H), 3.44 (s, 3H), 3.35-3.28 (m, 4H), 3.13-3.08 (m,2H).

LCMS: Retention Time=0.720 min.

1-9. (canceled)
 10. A method for the treatment of a warm-blooded animalhaving a bone-related disorder or condition, which is sensitive toinhibition of GSK-3 enzymes, which comprises administering to saidanimal an effective amount of a compound of the Formula I:

in which: n is 2 or 3; m is 2 or 3; R is methyl or ethyl; or apharmaceutically-acceptable salt thereof. 11-12. (canceled)
 13. A methodfor promoting bone formation in a warm-blooded animal, which comprisesadministering to said animal an effective amount of a compound of theFormula I

in which: n is 2 or 3; m is 2 or 3; R is methyl or ethyl; or apharmaceutically-acceptable salt thereof. 14-15. (canceled)
 16. A methodfor treating bone lesions in cancers such as for example cancers of thebreast, prostate and lung, multiple myeloma, osteosarcoma, Ewing'ssarcoma, chondrosarcoma, chordoma, malignant fibrous histiocytoma of thebone, fibrosarcoma of the bone, cancer induced bone disease, iatrogenicbone disease, benign bone disease and Paget's disease in a warm-bloodedanimal, which comprises administering to said animal an effective amountof a compound of the Formula I:

in which: n is 2 or 3; m is 2 or 3; R is methyl or ethyl; or apharmaceutically-acceptable salt thereof.
 17. (canceled)
 18. A methodaccording to claim 16, wherein the cancer is multiple myeloma.
 19. Amethod according to claim 10, 13 or 16, wherein the compound of formulaI is3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamide,or a pharmaceutically acceptable salt thereof.
 20. A method of claim 19,wherein the compound of formula I is a pharmaceutically acceptable saltof3-amino-6-(4-{3-[(3-methoxypropyl)amino]propyl}phenyl)-N-pyridin-3-ylpyrazine-2-carboxamide.21. A method of claim 20, wherein the pharmaceutically acceptable saltis a tri-hydrochloride, a mono-besylate or a mono-tosylate salt.